Bone Graft

ABSTRACT

An improved demineralized bone matrix (DBM) or other matrix composition is provided that has been mixed with a stabilizing agent that acts as (1) a diffusion barrier, (2) a enzyme inhibitor, (3) a competitive substrate, or (4) a masking moiety. A diffusion barrier acts as a barrier so as to protect the osteoinductive factors found in DBM from being degraded by proteolytic and glycolytic enzymes at the implantation site. Stabilizing agents may be any biodegradable material such as starches, modified starches, cellulose, dextran, polymers, proteins, and collagen. As the stabilizing agents degrades or dissolves in vivo, the osteoinductive factors such as TGF-.beta., BMP, and IGF are activated or exposed, and the activated factors work to recruit cells from the preivascular space to the site of injury and to cause differentiation into bone-forming cells. The invention also provides methods of preparing, testing, and using the inventive improved osteodinductive matrix compositions

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.10/271,140, filed on Oct. 15, 2002, which claims priority to U.S.Application Ser. No. 60/392,462, filed Jun. 27, 2002, and U.S.Application Ser. No. 60/329,156, filed Oct. 12, 2001, the contents ofwhich are incorporated herein by reference in its entirety.

BACKGROUND

The rapid and effective repair of bone defects caused by injury,disease, wounds, surgery, etc., has long been a goal of orthopaedicsurgery. Toward this end, a number of compositions and materials havebeen used or proposed for use in the repair of bone defects. Thebiological, physical, and mechanical properties of the compositions andmaterials are among the major factors influencing their suitability andperformance in various orthopaedic applications.

Autologous cancellous bone (“ACB”) is considered the gold standard forbone grafts. ACB is osteoinductive, is non-immunogenic and, bydefinition, has all of the appropriate structural and functionalcharacteristics appropriate for the particular recipient. Unfortunately,ACB is only available in a limited number of circumstances. Someindividuals lack ACB of appropriate dimensions and quality fortransplantation. Moreover, donor site morbidity can pose seriousproblems for patients and their physicians.

Much effort has been invested in the identification or development ofalternative bone graft materials. Demineralized bone matrix (“DBM”)implants have been reported to be particularly useful (see, for example,U.S. Pat. Nos. 4,394,370; 4,440,750; 4,485,097; 4,678,470; and4,743,259; Mulliken et al., Calcif. Tissue Int. 33:71, 1981; Neigel etal., Opthal. Plast. Reconstr. Surg. 12:108, 1996; Whiteman et al., J.Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin. Orthop. 293:360, 1993;each of which is incorporated herein by reference). Demineralized bonematrix is typically derived from cadavers. The bone is removedaseptically and/or treated to kill any infectious agents. The bone isthen particulated by milling or grinding and then the mineral componentis extracted (e.g., by soaking the bone in an acidic solution). Theremaining matrix is malleable and can be further processed and/or formedand shaped for implantation into a particular site in the recipient.Demineralized bone prepared in this manner contains a variety ofcomponents including proteins, glycoproteins, growth factors, andproteoglycans. Following implantation, the presence of DBM inducescellular recruitment to the site of injury. The recruited cells mayeventually differentiate into bone forming cells. Such recruitment ofcells leads to an increase in the rate of wound healing and, therefore,to faster recovery for the patient.

Current DBM formulations have various drawbacks. First, while thecollagen-based matrix of DBM is relatively stable, the active factorswithin the DBM matrix are rapidly degraded. The osteogenic activity ofthe DBM may be significantly degraded within 24 hours afterimplantation, and in some instances the osteogenic activity may beinactivated within 6 hours. Therefore, the factors associated with theDBM are only available to recruit cells to the site of injury for ashort time after transplantation. For much of the healing process, whichmay take weeks to months, the implanted material may provide little orno assistance in recruiting cells.

In addition to the active factors present within the DBM, the overallstructure of the DBM implant is also believed to contribute to the bonehealing capabilities of the implant.

SUMMARY OF THE INVENTION

The present invention provides improved demineralized bone matrix(“DBM”) compositions, related methods for preparing and using theinventive compositions, and kits containing the inventive compositions.The invention encompasses the recognition that the fast reduction inosteoinductive capabilities observed with previously available DBMcompositions may result from (1) degradation of osteoinductive agents,for example, as a result of proteases, sugar-degrading enzymes, or otherenzymes present in the host or the DBM itself, (2) diffusion ofosteoinductive agents out of the DBM; and/or (3) reduced activation ofosteoinductive agents in the DBM. The present invention thereforeprovides DBM compositions in which osteoinductive agents are protectedfrom degradation and/or from diffusion out of the composition. Thepresent invention may also include activation of the osteoinductivefactors found in the DBM, for example, in a controlled time releasemanner. In some embodiments, the invention also provides improvedshape-retaining characteristics contributing to the overall efficacy ofthe DBM compositions. Also, in some embodiments, the inventive DBMcomposition can be used as a delivery device to administer bioactiveagents.

Protection of the active factors within the DBM is provided using (1)diffusion barriers (e.g., polymers, starch), (2) enzyme inhibitors(e.g., protease inhibitors), (3) competitive substrates, and/or (4)masking moieties. Certain embodiments of the invention provide DBMcompositions comprising a stabilizing agent such as a polymer or otherfactor (e.g., protease inhibitors). Preferably, the polymer as adiffusion barrier is metabolized over time, so that the osteoinductiveagents are unmasked and/or released from the DBM composition over time,or retarded in their degradation rate. Diffusion barriers of theinvention may also work through alternative means by decreasing thediffusion of the activating enzymes to the factors present in the DBMcomposition. Preferably, such unmasking, release, controlled release, orcontrolled degradation occurs over a period longer than several hours,preferably longer than a day to several days, and possibly lasting weeksor even months. In certain preferred embodiments, the rates ofdegradation, release, and activation are balanced to yield a DBMcomposition with the desired level of osteoinductivity over time.Inventive compositions containing a stabilizing agent typically showosteoinductive activity for longer periods of time than is seen withcomparable compositions lacking the stabilizing agent.

In some embodiments of the invention, the stabilizing agent may comprisea polymer, such as a biodegradable polymer (e.g., that inhibits ordelays diffusion of osteoinductive agents out of the DBM composition,and/or blocks access of degrading and/or activating enzymes to theosteoinductive agents). Examples of biodegradable polymers includestarches, dextrans, cellulose, poly-esters, proteins, polycarbonates,polyarylates, and PLGA. Preferably the polymers are biocompatible andbiodegradable.

In other embodiments, inventive DBM compositions may include and/or betreated with agents that inhibit the activity of one or more activatingenzymes, proteases, or glycosidases. Such inhibitory agents are expectedto reduce the activity of specific enzymes (whether derived from thehost or from the DBM) that would otherwise interact with osteoinductiveagents or other active agents in the DBM, thereby reducingosteoinductivity or wound healing. Alternatively or additionally,inventive DBM compositions may include inhibitory agents presented in atime-release formulation (e.g., encapsulated in a biodegradablepolymer). In the case of activating enzymes (i.e., enzymes which lead tothe release, presentation, or creation of osteoinductive factors),inhibitory agents that reduce the activity of activating enzymespreferably lead to increased osteoinductivity over an extended period oftime rather than just a burst just after implantation.

Some embodiments of the present invention comprise DBM compositionsparticularly formulated to control or adjust the rate by which thecomposition, or portions thereof, lose osteoinductivity. To give but oneexample, DBM compositions may be prepared from multiple different DBMpreparations, each of which contains DBM particles of different sizeand/or including different amounts or types of stabilizing agents. Forinstance, DBM preparations or powders may be prepared that have varyinghalf-lives as determined by changing, for instance, the nature or amountof a stabilizing polymer, the extent of cross-linking of the polymer,the thickness of a stabilizing coating, the size of the particles, theamount of inhibitors of activating or degradatory enzymes, etc.Adjusting the amounts or locations of the different DBM preparationswithin the overall inventive DBM composition can modify thecharacteristics of part or all of the inventive composition. In thismanner, for example, the formulation could be customized to the patient,type of injury, site of injury, length of recovery, underlying disease,etc.

In another aspect, the present invention provides methods of preparinginventive improved DBM compositions. For instance, the present inventionprovides methods of formulating an improved DBM composition for aparticular site or injury.

The present invention also provides systems and reagents for preparingand applying DBM grafts, as well as systems and reagents for treatingbone defects using DBM implants. For example, the DBM composition may beprovided as a paste in a delivery device such as a syringe. Preferably,the DBM composition is sterile and is packaged so that it can be appliedunder sterile conditions (e.g., in an operating room).

The present invention further provides a system for characterizing DBMcomposites, and for identifying and preparing DBM-containing materialswith improved properties.

Furthermore, the present invention provides a system for deliveringbioactive agents, such as growth factors (e.g., bone morphogenicproteins, growth factors, hormones, angiogenic factors, cytokines,interleukins, osteopontin, osteonectin), to a host animal. The use of aDBM composition as a delivery vehicle for bioactive agents provides forthe unexpected result of an improved healing response to the implantwithout the need to administer separately the bioactive agent. A problemwith the introduction of the bioactive agent at the site is that it isoften diluted and redistributed during the healing process by thecirculatory systems (e.g., blood, lymph) of the recipient beforecomplete healing has occurred. A solution to this problem ofredistribution is to affix the bioactive components to the osteoimplant.Some preferred bioactive agents that can be delivered using a DBMcomposition include agents that promote the natural healing process,i.e., resorption, vascularization, angiogenesis, new growth, etc. A listof biological agents that may be delivered using inventive DBMcompositions is included as Appendix A. In preferred embodiments of thisaspect of the invention, an inventive composition is provided in whichDBM, together with a stabilizing agent, is used to deliver thebiologically active agent. It is expected that the stabilizing agentwill protect the biologically active agent from degradation, andtherefore will extend its active life after delivery into the recipientanimal. In certain embodiments, the bioactive agent is an osteoinductiveagent, and in certain embodiments, the DBM may be used to deliver morethan one bioactive agent, preferably more than two, and more preferablysometimes more than three bioactive agents. The bioactive agent may beassociated with the DBM. For example, the bioactive agent may beassociated with the DBM through electrostatic interactions, hydrogenbonding, pi stacking, hydrophobic interactions, van der Waalsinteractions, etc. In certain embodiments, the bioactive agent isattached to the DBM through specific interactions such as those betweena receptor and its ligand or between an antibody and its antigen. Inother embodiments, the bioactive agent is attached to the DBM throughnon-specific interactions (e.g., hydrophobic interactions).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Three week in-vivo radiographs showing evidence of boneformation.

FIG. 2. Six week x-rays or faxitron images.

FIG. 3. Qualitative evaluation of Vascularity (A) and ResidualDemineralized Bone Fiber (DBF) (B).

A. The vascularity and marrow cellularity increased on active DBF in adose-dependent fashion with increasing concentrations ofhrhBMP-2.times., which was not evident in the devitalized group. Thewild type rhBMP-2 at the 5 .mu.g dose was similar to the hybrid BMP.

B. The residual DBF remained a significant part of the nodule in each ofthe devitalized groups. The residual DBF dose-dependently deceased withincreasing amounts of hrhBMP-2.times. in the active DBF group. The wildtype rhBMP-2 was not as effective in remodeling the DBF as thehrhBMP-2.times.

FIG. 4. Comparison of untreated and hrhBMP-2.times. treated devitalizedand active DBF matrix.

Devitalized: Only residual DBF present with no bone formation elementsevident.

Devitalized+10 .mu.g hrhBMP-2.times.: New bone lining residual bone;extensive immature marrow with many adipocytes throughout nodule;extensive bone formation at outer edge of nodule but no rim present.

DBF: Rim of residual DBF present with extensive chondrocytes, bone, andsome marrow formation.

DBF+10 .mu.g hrhBMP-2.times.: Thin rim of mature new bone with extensivebone formation through out nodule with very little residual DBFremaining at center; extensively vascularized with well developedhematopoietic marrow present.

FIG. 5. Histological comparison of hrhBMP-2.times. and wild type rhBMP-2treated DBF matrix. There was significant bone formation in thehrhBMP-2.times. treated group compared to the rhBMP-2 group as evidencedby fewer spicules of bone and an extensive fatty marrow in the wild typegroup. A more developed, blood marrow was evident in the hybridrhBMP-2.times. group.

FIG. 6. Chemical structure of some examples of matrix metalloproteinaseinhibitors.

DEFINITIONS

Associated with: A stabilizing agent or other chemical entity isassociated with DBM or other osteogenic matrix according to the presentinvention if it is retained by the implant long enough to significantlyenhance the osteoinductivity of the implant. Specific examplesinclude 1) not freely diffusible from the DBM as determined in in vitrodiffusion assays in simulated body fluids; and/or 2) has an extendedhalf-life in the DBM as compared with free in solution. In someembodiments, associations are covalent; in others they are non-covalent.Examples of non-covalent interactions include electrostaticinteractions, hydrogen bonding, hydrophobic interactions, and van derWaals interactions. For instance, a bioactive agent may be renderedassociated with a DBM or other inventive matrix by virtue of a polymericstabilizing agent that restrains diffusion of the bioactive agent fromthe matrix. Alternatively or additionally, the bioactive agent may berendered associated with a DBM by virtue of a physical interaction withone or more entities that are themselves associated with the DBM. Forexample, the BMP-2 in Example 12 is considered to be associated with theDBM, and the BMP-2.times. is considered to be more closely associatedwith the DBM than the BMP-2.

Demineralized bone activity refers to the osteoinductive activity ofdemineralized bone.

Demineralized bone matrix, as used herein, refers to any materialgenerated by removing mineral material from living bone tissue. Inpreferred embodiments, the DBM compositions as used herein includepreparations containing less than 5% calcium and preferably less than 1%calcium by weight. Partially demineralized bone (e.g., preparations withgreater than 5% calcium by weight but containing less than 100% of theoriginal starting amount of calcium) are also considered within thescope of the invention.

Diffusion barrier refers to any material, coating, film, or substancethat decreases the rate of diffusion of a substance from one side of thebarrier to the other side, and more specifically, from outside to in orvice versa. The diffusion barrier in certain embodiments may be apolymer including proteins, polysaccharides, cellulose, man-madepolymer, PLGA, etc. that prevents the diffusion of activating agents(including water, enzymes, etc.) and/or degradatory enzymes into the DBMcomposition. The diffusion barrier may also prevent the movement ofosteoinductive factors out of the DBM composition. In certainembodiments, the diffusion barrier is biodegradable leading to thedegradation, activation, or release of osteoinductive factors over anextended period of time.

Matrix, as used herein, refers to a natural or non-natural substantiallysolid vehicle capable of association with at least one growth factor fordelivery to an implant site. The matrix may be completely insoluble ormay be slowly solubilized after implantation. Following implantation,preferred matrices resorb or degrade, remaining substantially intact forat least one to seven days, most preferably for two or four weeks orlonger and often longer than 60 days. Growth factors may be endogenouslypresent on the matrix as in the case of most demineralized bone, or theymay be exogenously added to the matrix. Matrices may also comprisecombinations of endogenous and exogenous growth factors. The matrix maybe in particulate or fiber form, or may be monolithic. The matrix maycomprise a number of materials and forms in combination such as fibersand particles. In one preferred embodiment, the matrix is comprised ofheat pressed demineralized bone fibers. In other embodiments, the matrixcomprises resorbable plastic polymers such as those described below assuitable for use as diffusion barriers. In other preferred embodiments,a particulated amorphous calcium phosphate is used as the matrix inassociation with an adsorbed growth factor such as a BMP. Morespecifically BMP-2 or BMP-4 or derivatives thereof. Still other matrixembodiments requiring the addition of an exogenous growth factorinclude, but are not limited to, particulated ceramics, preferablycalcium sulphates or calcium phosphates. The most preferred matrices arecalcium phosphates, the preparation of which is well known topractitioners in the art (see, for example, Driessens et al. “Calciumphosphate bone cements” Wise, D. L., Ed. Encyclopedic Handbook ofBiomaterials and Bioengineering, Part B, Applications New York: MarcelDecker; Elliott Structure and Chemistry of the Apatites and OtherCalcium Phosphates Elsevier, Amsterdam, 1994; each of which isincorporated herein by reference). Calcium phosphate matrices include,but are not limited to, dicalcium phosphate dihydrate, monetite,tricalcium phosphate, tetracalcium phosphate, hydroxyapatite,nanocrystalline hydroxyapatite, poorly crystalline hydroxyapatite,substituted hydroxyapatite, and calcium deficient hydroxyapatites.

Osteoinductive, as used herein, refers to the quality of being able tostimulate bone formation. Any material that can induce the formation ofectopic bone in the soft tissue of an animal is consideredosteoinductive. For example, most osteoinductive materials induce boneformation in athymic rats when assayed according to the method ofEdwards et al. (“Osteoinduction of Human Demineralized Bone:Characterization in a Rat Model” Clinical Orthopeadics & Rel. Res.,357:219-228, December 1998; incorporated herein by reference).Osteoinductivity in some instances is considered to occur throughcellular recruitment and induction of the recruited cells to anosteogenic phenotype. Osteoinductivity may also be determined in tissueculture as the ability to induce an osteogenic phenotype in culturecells (primary, secondary, or explants) It is advisable to calibrate thetissue culture method with an in vivo ectopic bone formation assay asdescribed by Zhang et al. “A quantitative assessment of osteoinductivityof human demineralized bone matrix” J. Periodontol. 68(11): 1076-84,November 1997; incorporated herein by reference. Calibration of the invitro assays against a proven in vivo ectopic bone formation model iscritical because the ability of a compound to induce an apparent“osteogenic” phenotype in tissue culture may not always be correlatedwith the induction of new bone formation in vivo. BMP, IGF, TGF-.beta.,parathyroid hormone (PTH), and angiogenic factors are only some of theosteoinductive factors found to recruit cells from the marrow orperivascular space to the site of injury and then cause thedifferentiation of these recruited cells down a line responsible forbone formation. DBM isolated from either bone or dentin have both beenfound to be osteoinductive materials (Ray et al., “Bone implants” J.Bone Joint Surgery 39A:1119, 1957; Urist, “Bone: formation byautoinduction” Science 150:893, 1965; each of which is incorporatedherein by reference).

Osteoinductivity score refers to a score ranging from 0 to 4 asdetermined according to the method of Edwards et al. (1998) or anequivalent calibrated test. In the method of Edwards et al., a score of“0.infin. represents no new bone formation; “1” represents 1%-25% ofimplant involved in new bone formation; “2” represents 26-50% of implantinvolved in new bone formation; “3” represents 51%-75% of implantinvolved in new bone formation; and “4” represents >75% of implantinvolved in new bone formation. In most instances, the score is assessed28 days after implantation. However, for the improved inventiveformulations, particularly those with osteoinductivity comparable to theBMPs, the osteoinductive score may be obtained at earlier time pointssuch as 7, 14, or 21 days following implantation. In these instances itis important to include a normal DBM control such as DBM powder withouta carrier, and if possible, a positive control such as BMP. Occasionallyosteoinductivity may also be scored at later timepoints such as 40, 60,or even 100 days following implantation. Percentage of osteoinductivityrefers to an osteoinductivity score at a given time point expressed as apercentage of activity, of a specified reference score.

Particle or fibers refers to a preparation of DBM, DBM compositions, orbone sample that has been milled, ground, pulverized, or otherwisereduced to a particulate form. The size of the particles or fibers istypically greater than 50 microns, preferably greater than 75 microns,more preferably greater than 100 microns, and most preferably greaterthan 150 microns. These dimensions refer to average particle diameterfor more spherical-like particles, and for particles of other shapesexcept where specifically indicated it refers to the smallestcross-sectional dimension of the particle. In certain embodiments, thecomposition may include even larger sized particles, preferably greaterthan 1 mm, greater than 1.5 mm, or most preferably greater than 2 mm intheir largest dimension. The particles or fibers may be of any shapeincluding wedges, rods, spheres, cubes, discs, ovals, irregularlyshaped, etc. For example, in certain embodiments, the particles may bewedge-shaped and be approximately 2 mm in their largest dimension and100 microns or less in another dimension. The particles or fibers may besieved or sorted in order to collect particles of a particular size.These particles or fibers may be mixed with a solution, slurry,deformable solid, or liquid to form a paste to be used in administeringor applying the graft of DBM, inventive DBM composition, or bone sample.Preferred methods of particle or fiber preparation are disclosed inissued U.S. Pat. Nos. 5,607,269; 5,236,456; 5,284,655; 5,314,476; and5,507,813; each of which is incorporated herein by reference.

Polysaccharide, as used herein, refers to any polymer or oligomer ofcarbohydrate residues. The polymer may consist of anywhere from two tohundreds to thousands of sugar units. Polysaccharides may be purifiedfrom natural sources such as plants or may be synthesized de novo in thelaboratory. Polysaccharides isolated from natural sources may bemodified chemically to change their chemical or physical properties(e.g., phosphorylated, cross-linked). Polysaccharides may also be eitherstraight or branch-chained. They may contain both natural and/orunnatural carbohydrate residues. The linkage between the residues may bethe typical ether linkage found in nature or may be a linkage onlyavailable to synthetic chemists. Examples of polysaccharides includecellulose, maltin, maltose, starch, modified starch, dextran, andfructose. Glycosaminoglycans are also considered polysaccharides.

Protease inhibitors, as used herein, are chemical compounds capable ofinhibiting the enzymatic activity of protein cleaving enzymes (i.e.,proteases). The proteases inhibited by these compounds include serineproteases, acid proteases, metalloproteases (examples of some matrixmetalloprotease inhibitors are shown in FIG. 6), carboxypeptidase,aminopeptidase, cysteine protease, etc. The protease inhibitor may actspecifically to inhibit only a specific protease or class of proteases,or it may act more generally by inhibiting most if not all proteases.Preferred protease inhibitors are protein or peptide based and arecommercially available from chemical companies such as Aldrich-Sigma.Protein or peptide-based inhibitors which adhere to the DBM (or calciumphosphate or ceramic carrier) are particularly preferred as they remainassociated with the matrix providing a stabilizing effect for a longerperiod of time than freely diffusible inhibitors. Examples of proteaseinhibitors include aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride(AEBSF), amastatin-HCl, alpha1-antichymotrypsin, antithrombin III,alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF),arphamenine A, arphamenine B, E-64, bestatin, CA-074, CA-074-Me, calpaininhibitor I, calpain inhibitor II, cathepsin inhibitor, chymostatin,diisopropylfluorophosphate (DFP), dipeptidylpeptidase IV inhibitor,diprotin A, E-64c, E-64d, E-64, ebelactone A, ebelactone B, EGTA,elastatinal, foroxymithine, hirudin, leuhistin, leupeptin,alpha2-macroglobulin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A,phebestin, 1,10-phenanthroline, phosphoramidon, chymostatin, benzamidineHCl, antipain, epsilon-aminocaproic acid, N-ethylmaleimide, trypsininhibitor, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor, andsodium EDTA.

A peptide or protein, according to the present invention, comprises astring of at least two amino acids linked together by peptide bonds.Inventive peptides preferably contain only natural amino acids, althoughnon-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain) and/or amino acidanalogs as are known in the art may alternatively be employed. Also, oneor more of the amino acids in an inventive peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc.

Stabilizing agent is any chemical entity that, when included in aninventive composition comprising DBM and/or a growth factor, enhancesthe osteoinductivity of the composition as measured against a specifiedreference sample. In most cases, the reference sample will not containthe stabilizing agent, but in all other respects will be the same as thecomposition with stabilizing agent. The stabilizing agent also generallyhas little or no osteoinductivity of its own and works either byincreasing the half-life of one or more of the active entities withinthe inventive composition as compared with an otherwise identicalcomposition lacking the stabilizing agent, or by prolonging or delayingthe release of an active factor. In certain embodiments, the stabilizingagent may act by providing a barrier between proteases andsugar-degrading enzymes thereby protecting the osteoinductive factorsfound in or on the matrix from degradation and/or release. In otherembodiments, the stabilizing agent may be a chemical compound thatinhibits the activity of proteases or sugar-degrading enzymes. In apreferred embodiment, the stabilizing agent retards the access ofenzymes known to release and solubilize the active factors. Half-lifemay be determined by immunological or enzymatic assay of a specificfactor, either as attached to the matrix or extracted there from.Alternatively, measurement of an increase in osteoinductivity half-life,or measurement of the enhanced appearance of products of theosteoinductive process (e.g., bone, cartilage or osteogenic cells,products or indicators thereof) is a useful indicator of stabilizingeffects for an enhanced osteoinductive matrix composition. Themeasurement of prolonged or delayed appearance of a strongosteoinductive response will generally be indicative of an increase instability of a factor coupled with a delayed unmasking of the factoractivity.

Targeting agent is any chemical entity that, when included in aninventive compositions, will direct the composition to a particular siteor cause the inventive composition to remain in a particular site withinthe recipient's body. A targeting agent may be a small molecule,peptide, protein, biological molecule, polynucleotide, etc. Typicaltargeting agents are antibodies, ligands of known receptors, andreceptors. These targeting agents may be associated with the inventivecomposition through covalent or non-covalent interactions so that theinventive composition is directed to a particular tissue, organ, injuredsite, or cell type.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

As described herein, the present invention provides compositions andmethods relating to improved DBM or synthetic growth factor containingcompositions. Below, certain aspects of preferred embodiments of theinvention are described in more detail and with reference to the Figuresof the Drawing. Those of ordinary skill will appreciate that a varietyof embodiments or versions of the invention are not specificallydiscussed below but are nonetheless within the scope of the presentinvention, as defined by the appended claims.

DBM is comprised principally of proteins and glycoproteins, collagenbeing the primary protein substituent of DBM. While collagen isrelatively stable, being degraded only by the relatively rarecollagenase enzymes, the other proteins and active factors present inDBM are quickly degraded by enzymes present in the host. Thesehost-derived enzymes include proteases and sugar-degrading enzymes(e.g., endo- and exo-glycosidases, glycanases, glycolases, amylase,pectinases, galacatosidases, etc.). Many of the active growth factorsresponsible for the osteoinductive activity of DBM exist in crypticform, in the matrix until activated. Activation can involve the changeof a pre or pro function of the factor, or release of the function froma second factor or entity which binds to the first growth factor. Theinstant invention alters the time course over which the active factorspresent in DBM can exert their osteoinductive activity either by 1)slowing the degradation of the active factors present in DBM, therebyallowing them longer residence time as active moieties, or 2) prolongingthe release of one or more active factors from the implant, or 3)altering the kinetics of activation of one or more cryptic factors. Theinstant invention increases the effective osteoinductivity of the DBMcomposition by (1) altering the kinetics of activation of crypticfactors, (2) altering the delivery and/or release of active factor fromthe matrix, and/or (3) reducing proteolytic degradation of the activefactor within or as they are released from the DBM composition.Increased bone formation presumably occurs through the recruitment ofmore cells into the osteogenic phenotype.

The instant invention provides four approaches to the protection ofactive factors from degradation by either host-derived or endogenousenzymes. Factors to be protected may be endogenous to DBM preparationsor factors added to either DBM or synthetic matrix compositions.Protection is provided through the use of a) diffusion barriers, b)enzyme inhibitors, c) competitive substrates, and/or d) maskingmoieties. These same four approaches may be used to control theactivation and/or release of osteoinductive factors in cryptic form. Forexample, diffusion barriers or activating enzyme inhibitors preventactivating enzyme from reaching the cryptic factors or from acting uponthe cryptic factors. Preferably, degradation, release, and activation ofactive factors within the DBM composition is balanced to yield a desiredosteoinductivity profile over time.

Demineralized Bone Matrix

DBM preparations have been used for many years in orthopaedic medicineto promote the formation of bone. For example, DBM has found use in therepair of fractures, in the fusion of vertebrae, in joint replacementsurgery, and in treating bone destruction due to underlying disease suchas rheumatoid arthritis. DBM is thought to promote bone formation invivo by osteoconductive and osteoinductive processes. Osteoconductionoccurs if the implanted material serves as a scaffold for the support ofnew bone growth. Osteoconduction is particularly significant when bonegrowth is desired across a large or “critical size” defect, across whichbone healing would proceed only slowly or not at all. It is generallybelieved that the osteoconductive properties of DBM preparations areprovided by the actual shape and coherence of the implant. Thus DBMcompositions comprising entangled fibers tend to have superiorosteoconductive properties as compared to less fibrous, more granularpreparations. Stabilizing agents which tend to preserve the shape and/orcoherence of the DBM substituent can lead to better bone formingproperties.

The osteoinductive effect of implanted DBM compositions is thought toresult from the presence of active growth factors present on theisolated collagen-based matrix. These factors include members of theTGF-.beta., IGF, and BMP protein families. Particular examples ofosteoinductive factors include TGF-.beta., IGF-1, IGF-2, BMP-2, BMP-7,parathyroid hormone (PTH), and angiogenic factors. Other osteoinductivefactors such as osteocalcin and osteopontin are also likely to bepresent in DBM preparations as well. There are also likely to be otherunnamed or undiscovered osteoinductive factors present in DBM.

Any of a variety of demineralized bone matrix preparations may beutilized in the practice of the present invention. DBM prepared by anymethod may be employed including particulate or fiber-basedpreparations, mixtures of fiber and particulate preparations, fully orpartially demineralized preparations, mixtures of fully and partiallydemineralized preparations, including surface demineralized preparationsas described by Gertzman et al. (U.S. Pat. No. 6,326,018, issued Dec. 4,2001; incorporated herein by reference). Preferred DBM compositions aredescribed by Dowd et al., U.S. Pat. No. 5,507,813, which is incorporatedherein by reference. Also useful are DBM preparations comprisingadditives or carriers such as polyhydroxyl compounds, polysaccharides,glycosaminoglycan proteins, nucleic acids, polymers, polaxomers, resins,clays, calcium salts, and/or derivatives thereof.

In certain embodiments, the DBM material utilized to formulate inventivecompositions has greater than 50%, preferably greater than 75%, morepreferably greater than 80%, 85%, 90%, or 95% and most preferablygreater than 98% of the calcium phosphate removed. The bone used increating the DBM may be obtained from any source of living or deadtissue. Often, it will be preferred that the source of bone be matchedto the eventual recipient of the inventive composition. At a minimum, itis often desirable that the donor and recipient are of the same species,though even xenogenic sources are permitted.

Once a bone sample is obtained, it is milled, ground, pulverized, orotherwise reduced to particulate form. In preferred embodiments, theparticles will be greater than 75 microns in their minimum dimension,more preferably greater than 100 microns, and more preferably greaterthan 150 microns. However, it should be noted that one method of thepreferred invention is to stabilize implants containing particles lessthan 100 microns in any dimension and potentially even less than 75microns. Particles of 75 microns or less, following demineralization,are known to have limited or no osteoinductivity, and aspects of thepresent invention may be used to enhance the activity of these smallsize particles as well. For preparations employing DBM of these smallsizes, at least one stabilizing agent is used which retards the influxof host cells capable of removing such small particles (e.g.,macrophages and foreign body giant cells) long enough to allow theactive factors within the DBM to elicit an osteoinductive response. Inaddition or alternatively, a diffusion barrier will be present to retardthe efflux of factors from the particles. In certain embodiments, theparticles are at least 200 microns across the greatest dimension. Theparticles may be any shape including ovals, spherical, cuboidal, cones,pyramids, wedges, etc. In certain embodiments, the particles are wedges,pyramids, or cones being 200 microns across their largest dimension. Inother embodiments, the DBM composition may include a mixture of severaldifferent sizes and/or shapes of particles.

Following particulation, the DBM is treated to remove mineral from thebone. While hydrochloric acid is the industry-recognizeddemineralization agent of choice, the literature contains numerousreports of methods for preparing DBM (see, for example, Russell et al.Orthopaedics 22(5):524-531, May 1999; incorporated herein by reference).For the purposes of the present invention, any material that provides ascaffolding containing active osteoinductive factors is considered DBM.The DBM may be prepared by methods known in the art or by other methodsthat can be developed by those of ordinary skill in the art withoutundue experimentation. In some instances, large fragments or even wholebone may be demineralized, and then particulated followingdemineralization. DBM prepared in this way is within the scope of theinvention.

In the preparing the improved DBM compositions, the DBM component may beground or otherwise processed into particles of an appropriate sizebefore or after demineralization. In certain embodiments, the particlesize is greater than 75 microns, more preferably ranging from about 100to about 3000 microns, and most preferably from about 200 to about 2000microns. After grinding the DBM component to the desired size, themixture may be sieved to select those particles of a desired size. Incertain embodiments, the DBM particles may be sieved though a 50 micronsieve, more preferably a 75 micron sieve, and most preferably a 100micron sieve.

One particularly useful way to protect small size particles fromcellular ingestion and/or provide a diffusion barrier is to embed themin a monolithic bioabsorbable matrix, and then fragment theparticle-containing monolithic matrix into particle sizes greater than70 microns, preferably greater than 100 microns, and most preferablygreater than 150 microns in their smallest dimension. Preferred matricesfor embedding small DBM particles include biocompatible polymers andsetting calcium phosphate cements. Generally the particulate DBM/polymerweight ratio will range from about 1:5 to about 1:3. In the case ofcalcium phosphate, the DBM will be present up to 75% by weight.Particulation of the monolith can be accomplished by conventionalmilling or grinding, or through the use of cryomilling, or freezingfollowed by pulverization. In one preferred embodiment, lyophilized DBMis embedded in a resorbable polymer. In a second preferred embodiment,lyophilized DBM is embedded in one of the setting calcium phosphatesknown to the art.

Stabilizing Agents

Diffusion barriers. Diffusion barriers retard the diffusion ofdegradative enzymes and/or water to the active moieties within theinventive formulations. Enzymes retarded in their diffusion to theincluded DBM may be capable of releasing the active factor from thematrix, and/or degrading or inactivating the active factor. They alsomay act by retarding diffusion of the active factors from the implantsite. In these ways, the barriers provide for longer residence time ofthe active factors at the implant site. This is particularly useful forforming bone in higher species such as humans, where bone formationappears to require the presence of active factors for longer times.

Generally, materials most suitable to serve as diffusion barriers willbe easily mixed with DBM or synthetic matrix of choice to form a gel,paste, or putty-like consistency, although in some embodiments, thebarrier/matrix formulation will be prepared as a relativelynon-deformable solid (e.g., for matrix preparations to be used inposterior lateral spine fusion). In preferred embodiments, the diffusionbarriers themselves degrade in a predictable manner to unmask activefactors at a time later than would normally occur in the absence of adiffusion barrier. Resorbable polymers with known hydrolytic rates areuseful as diffusion barriers as well as enzymatically degraded polymers.Particularly useful are lipase susceptible lipid based carriers such asfatty acids and phospholipids, which mix well with DBM. In certain DBMembodiments, the composition does not include phosphatidylcholine. Someparticularly effective preparations provide prolonged stability bycontrolled unmasking of the osteoinductive factors. These preparationsgenerally involve the use of two or more diffusion barriers withdifferent degradation times affording at least two different rates ofunmasking the same active factor.

Biodegradable polymers useful in preparing inventive stabilizedmatrix/growth factor compositions include natural polymers such asproteins (e.g., collagen) and polysaccharides (e.g., starch, modifiedstarch, maltrin) as well as man-made resorbable polymers such aspoly-orthoesters. These polymers when mixed with the inventive growthfactor containing compositions retard diffusion of the host'sdegradative enzymes and/or water to the active factors contained withinthe composition, thereby retarding release and/or degrading of theactive factor contained therein.

Polymers that may be included within inventive compositions include, forexample, natural polymers such as lipids, polysaccharides,proteoglycans, and proteins. Preferred polysaccharides include starches,dextrans, and celluloses, and preferred proteins include collagen.Polysaccharides such as starches, dextrans, and celluloses may beunmodified or may be modified physically or chemically to affect one ormore of their properties such as their characteristics in the hydratedstate, their solubility, their susceptibility to degradation, or theirhalf-life in vivo. Polysaccharides such as starches and celluloses areattractive as they also have known degradation rates. Generally, thecelluloses degrade more slowly within the body, breaking down on theorder of weeks or months, while many starch and lipid preparationsdegrade rapidly, on the order of hours or days. Starch in the naturalstate is a mixture of two polysaccharides, amylose and amylopectin. Thesusceptibility of the particular starch to the starch-degrading enzymessuch as amylase, pectinases, and .beta.-glucosidase is an importantconsideration in designing the inventive formulations. Those skilled inthe art are aware of the variety of amylase susceptibilities of starchesprepared from various plant sources and may apply this knowledge toproduce formulations having a desired stability time. Preferred starcheswill degrade as much as 10% per day, preferably 50% per day, and mostpreferably greater than 90% per day. Those starches less susceptible todegradation by pectinase and/or amylase (amylase-resistant starch;Starch Australasia, Sydney, Australia) may be used to maximally extendthe osteoinductive half-life in vivo to an even greater extent thanimproved DBM or synthetic growth factor/matrix formulations preparedfrom more enzyme susceptible starches. Some modified starches are lesssusceptible to degradation by amylase; therefore, improved DBM withmodified starch would presumably have a longer half-life in vivo ascompared to those improved DBM with unmodified starch. One preferredmethod to affect amylase susceptibility of starch is through the use ofstarch lipid combinations. Guidance for the combination of lipid andstarch to affect amylase susceptibility is given by Crowe et al“Inhibition of Enzymic Digestion of Amylose by Free Fatty Acids In VitroContributes to Resistant Starch Formation” J. Nutr. 130(8):2006-2008,August 2000; incorporated herein by reference. Similar considerationsapply to lipids and their degradative enzymes the lipases. A largevariety of mono-, di-, and triglycerides with varying degrees ofsusceptibility to lipase degradation are available from commercialsources. Some embodiments include one or more polymeric materials,preferably biodegradable, such as tyrosine polycarbonates,polyfumarates, tyrosine polyarylates, and poly-orthoesters such aspolylactide, polygalactide, and co-polymers thereof. These polymers arebiodegradable, and their properties can be modified by altering thechain length or degree of cross-linking of the polymer and/or thechemical structure of the monomers. Additionally, co-polymers can beprepared using combinations of resorbable polymers.

Enzyme inhibitors. Alternatively or additionally, the inventivecompositions may be stabilized by the addition of one or moredegradation inhibitors, active against growth factor activity degradingagents found in the host organism and/or in the implant composition.These inhibitors may also inhibit the activity of enzymes responsibleactivating osteoinductive factors of the DBM composition. Degradation oractivation inhibitors useful in the practice of the present inventionmay include, for example, acid protease inhibitors, serine proteaseinhibitors, metalloprotease inhibitors (shown in FIG. 6; also, seeWhittaker et al. “Matrix Metalloproteinases and their Inhibitors-CurrentStatus and Future Challenges” Celltransmissions 17(1):3-14; incorporatedherein by reference), cysteine protease inhibitors, glyconaseinhibitors, and glycosidase inhibitors. Specific protease inhibitorsuseful in the practice of the present invention include, for example,aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF),amastatin-HCl, alpha1-antichymotrypsin, antithrombin III,alpha1-antitrypsin, 4-aminophenylmethane sulfonyl-fluoride (APMSF),arphamenine A, arphamenine B, E-64, bestatin, CA-074, CA-074-Me, calpaininhibitor I, calpain inhibitor II, cathepsin inhibitor, chymostatin,diisopropylfluorophosphate (DFP), dipeptidylpeptidase IV inhibitor,diprotin A, E-64c, E-64d, E-64, ebelactone A, ebelactone B, EGTA,elastatinal, foroxymithine, hirudin, leuhistin, leupeptin,alpha2-macroglobulin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A,phebestin, 1,10-phenanthroline, phosphoramidon, chymostatin, benzamidineHCl, antipain, epsilon-aminocaproic acid, N-ethylmaleimide, trypsininhibitor, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK),1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK), trypsin inhibitor,sodium EDTA, and the TIMPs class of metalloproteinase inhibitors.Particularly useful ones are those stable under acidic conditions andeffective at acidic conditions. As will be appreciated by those of skillin this art, the less osteoinductive factors lost or degraded during theprocessing of the bone to form DBM the more will be available forrecruitment once the DBM composition is implanted.

In some embodiments, a composition for implantation is providedcomprising demineralized bone matrix and an excipient or stabilizingagent. Such composition for implantation may exhibit an osteoinductivitythat is at least about 10% greater than osteoinductivity of acomposition of demineralized bone matrix without excipient. Theexcipient may be, for example, a diffusion barrier, an enzyme inhibitor,a competitive substrate, a masking entity, a polymer (natural,non-natural, modified, or derivated), growth factor binding protein,lectin, antibody, other material, and combinations thereof. Varioussuitable excipients are discussed more fully below. These excipients areintended to be illustrative only. Further, in some embodiments, thedemineralized bone matrix may be embedded in the excipient.

Competitive substrates. Use of competitive substrates for the host'sdegradative or activating enzymes may also be employed to stabilize theosteoinductive factors of the DBM or exogenously added growth factors.Examples of competitive substrates include di- and poly-lysines. Di- andpolysaccharides can be employed as competitive substrates ofglycosidases, amylases, and/or pectinases. Particularly useful arestereoisomers of the competitive substrates.

Masking entities. Specific masking entities are generally used tospecifically block a single entity or class of entities from enzymaticbreakdown. The degradative or activating enzyme to be blocked may beendogenous or exogenous to the matrix. The masking entities generallybind to a ligand present on the matrix which may or may not be theactive factor itself. Once bound the masking entity sterically hindersthe breakdown and/or release of one or more active factors. Over timethe masking entity either unbinds or itself is degraded leaving theligand and or growth factor susceptible to degradation. Diffusionbarriers represent a generalized form of masking entity by preventingaccess of the degradative or activating enzymes to many or all thegrowth factors associated with the matrix.

Growth factor binding proteins: Virtually every extracellular matrixgrowth factor is know to be associated with a binding protein whichregulates the activity of the growth factor. Purified preparations ofthese binding proteins can be prepared, and added to DBM preparations toserve as masking entities. Typical growth factor binding proteinsinclude but are not limited to noggin, chordin, follistatin, TGF-.beta.binding protein, and insulin-like growth factor binding proteins. Agentsmay also be added to the DBM composition to induce the release of thegrowth factor from its binding protein. In certain embodiments, theagent known to induce release of the growth factor may be encapsulatedin a biodegradable polymer so that the agent is released over anextended period of time, thereby leading to the release of growth factorover an extended period of time.

Lectins. Lectins are proteins which can bind to the sugar moieties ofglycoproteins. Since growth factors are generally glycoproteins, lectinscan be employed to bind to the growth factors and potentially retard orinhibit access of proteases or growth factor releasing enzymes to theactive growth factors. Ideally the lectin will be selected according tothe identity of the terminal sugar on the active glycoprotein ofinterest. Lectins include, but are not limited to, membrane-boundlectins, I-type lectins, and P-type lectins. Specific lectins includegalectins, calcium-dependent lectins, selecting, collecting, andannexins.

Antibodies. Monoclonal or polyclonal antibodies specific to the activefactors, or to those proteins known to bind to the active factors (seeabove) may be added to the inventive formulations to protect specificgrowth factors from degradative or releasing enzymes.

Inventive DBM compositions may alternatively or additionally bestabilized through exposure to conditions (e.g., pH, temperature, etc.)under which degrading agents do not function optimally or thedegradatory enzymes will not function effectively (e.g., low pH).

Addition of enzyme inhibitors, competitive substrates, and maskingagents. The incorporation of any of these entities into the inventiveformulations, is generally accomplished by suspending the molecule ormolecules of interest in an appropriately compatible buffer as will beknown to those skilled in the art. This buffer will be mixed withlyophilized matrix in a relatively low liquid-to-solid volume ratio toform a slurry. The slurry is then lyophilized and used to prepare thedesired DBM formulations.

One unexpected feature of the instant invention is that theincorporation of any of the inventive enzyme inhibitors, competitivesubstrates, or masking agents often has the additional feature ofimproving the DBM formulation shelf-life by preventing access ofendogenously present degradative enzymes to the active factors presentin the matrix. This is particularly true for DBM formulations which areprepared containing water (e.g., DBM preparations with hydrogel carrierssuch as hyaluronic acid or collagen, or hydrated starch carriers).

Many of the osteoinductive factors found in DBM are in cryptic form andmust be “activated” or “released” in order to be osteoinductive. Theactivation of osteoinductive factors may involve a conformationalchange, a post-translational modification, a cleavage of the peptide, achange in tertiary or quaternary structure, release from the DBM,release from a binding protein, etc. For example, the factors may be ina pre- or pro-form which requires proteolytic cleavage to be active. Inaddition, the osteoinductive factors may be associated with a bindingprotein or a protein of the matrix of the DBM. The same processes suchas proteolysis involved in degradation of the active factors may also beinvolved in the activation of these factors. Therefore, all the samemethods described above that can be used to slow degradation may alsoaffect activation rates. One of skill in the art preparing a DBMcomposition could balance the rates of degradation and activation toachieve a desired level of osteoinductivity from the implant over time.In addition, such factors as pH, ion concentration, or other factorswhich affect protein function and/or folding may affect the activationof osteoinductive factors found in DBM. These factors also may effectthe release of a factor from its binding protein. In certainembodiments, for example, where pH plays a role in the activation of afactor, the DBM composition may include a chemical compound such as apolymer which will break down over time and release an acid by-product;thereby, activating the factors within the DBM composition. In otherembodiments, a biodegradable polymer may release ions or a protease thatis able to “activate” the osteoinductive factors of the DBM composition.

Release of the osteoinductive factors from the delivery matrix may alsobe important in its osteoinductivity. Many factors may be found bound tothe DBM through specific binding proteins as described above or throughnon-specific interactions. A portion of the factors may need to bereleased from the matrix in order to be active while others may only beactive while bound. For example, cells may be recruited to the matrix bycertain factors, and then once there, the cells may interact with otherfactors bound to the matrix. The cells may need to interact with boththe matrix and the factor to induce bone production. The rate of releaseof the osteoinductive factors may be controlled by diffusion barriers oragents which affect the binding of the factors to the matrix or theirbinding proteins. As described above, in certain embodiments, it ispreferred that a diffusion barrier be degraded over time so as torelease factors or allow recruited cells to interact with the matrix.Degradation of the diffusion barrier may also allow proteases into theDBM implant to activate and/or release osteoinductive factors.

As will be appreciated by one of skill in this art, the DBM compositionmay be prepared to balance degradation, activation, and release ofosteoinductive factors to create a composition with a desiredosteoinductive activity. The osteoinductivity of the DBM composition maybe suited for a particular application, site of implant, or patient. Forinstance, certain application would require an extended period ofosteoinductivity ranging from weeks to months; whereas otherapplications may only need osteoinductivity for days to weeks. One ofskill in the art can prepare a DBM composition with a desiredosteoinductivity time profile.

Test for Enhancement

The invention also provides a simple in vitro test for the screening ofsuitable stabilizing agents. DBM prepared with and without thebiodegradable stabilizing agent is exposed under simulated physiologicalconditions (e.g., pH 7.4, physiological saline) to an enzyme orcombination of enzymes known to be capable of degrading some or all ofthe protein constituents of the DBM. Most often this will be a proteasesuch as trypsin, papain, peptidase, or the like. Evidence for matrix ormatrix component breakdown is compared between the two preparations.Materials retarding the breakdown process are considered to be goodcandidates for further testing. Preferred indicators of breakdowninclude immunological detection of TGF-.beta. and/or IGF breakdown. Inaddition to the enzymes indicated above, other enzymes such ascollagenases or combinations of enzymes as well as glycosidases may alsobe used. Particularly useful in this regard is the natural degradatoryactivity of serum or tissue extracts. Under these conditions, specificmarker proteins present in the DBM may be tracked by immunologicalmethods such as radioimmunoassay or gel electrophoresis utilizingwestern blots, or other analytical methods known in the art.

Following the identification of candidate stabilizers in the aboveassay, the DBM formulations containing the candidate stabilizers aretested in the osteoinductivity assays described elsewhere herein.

Osteoinducer

To the improved DBM may be added other osteoinducing agents. Theseagents may be added in an activated or non-activated form. These agentsmay be added at anytime during the preparation of the inventivematerial. For example, the osteoinducing agent may be added after thedemineralization step and prior to the addition of the stabilizingagents so that the added osteoinducing agent is protected from exogenousdegrading enzymes once implanted. In some embodiments the DBM islyophilized in a solution containing the osteoinducing agent. In certainother preferred embodiments, the osteoinducing agents are adhered ontothe hydrated demineralized bone matrix and are not freely soluble. Inother instances, the osteoinducing agent is added to the improved DBMafter addition of the stabilizing agent so that the osteoinducing agentis available immediately upon implantation of the DBM.

Osteoinducing agents include any agent that leads to or enhances theformation of bone. The osteoinducing agent may do this in any manner,for example, the agent may lead to the recruitment of cells responsiblefor bone formation, the agent may lead to the secretion of matrix whichmay subsequently undergo mineralization, the agent may lead to thedecreased resorption of bone, etc. Particularly preferred osteoinducingagents include bone morphogenic proteins (BMPs), transforming growthfactor (TGF-.beta.), insulin-like growth factor (IGF-1), parathyroidhormone (PTH), and angiogenic factors such as VEGF. In one preferredembodiment (Example 12), the inducing agent is genetically engineered tocomprise an amino acid sequence which promotes the binding of theinducing agent to the DBM or the carrier. Sebald et al. inPCT/EP00/00637, incorporated herein by reference, describe theproduction of exemplary engineered growth factors, suitable for use withDBM.

Formulation

Improved osteogenic compositions of the present invention may beformulated for a particular use. The formulation may be used to alterthe physical, biological, or chemical properties of a DBM preparation. Aphysician would readily be able to determine the formulation needed fora particular application taking into account such factors as the type ofinjury, the site of injury, the patient's health, the risk of infection,etc.

Inventive compositions therefore may be prepared to have selectedresorption/loss of osteoinductivity rates, or even to have differentrates in different portions of an implant. For example, the formulationprocess may include the selection of DBM particles of a particular sizeor composition, combined with the selection of a particular stabilizingagent or agents, and the amounts of such agents. To give but oneexample, it may be desirable to provide a composition whoseosteoinductive factors are active in a relatively constant amount over agiven period of time. A DBM composition comprising factors with longerhalf-lives can be prepared using a less biodegradable polymer or alarger amount (e.g., a thicker coating) of polymeric compound.Alternatively or additionally, the particle size may be important indetermining the half-life of the inventive DBM composition. In certainpreferred embodiments, an inventive formulation may include a mixture ofparticles, each with a different half-life. Such a mixture could providethe steady or possible unmasking of osteoinductive factors over anextended period of time ranging from days to weeks to months dependingon the needs of the injury. Compositions such as this can be formulatedto stimulate bone growth in a human patient comparable to the bonegrowth induced by treatment with 10 .mu.g of rhBMP on a collagen sponge,and preferably comparable to 100 .mu.g, and most preferably 1-10 mgrhBMP.

Physical properties such as deformability and viscosity of the DBM mayalso be chosen depending on the particular clinical application. Theparticles of the improved DBM may be mixed with other materials andfactors to improve other characteristics of the implant. For example,the improved DBM material may be mixed with other agents to improvewound healing. These agents may include drugs, proteins, peptides,polynucleotides, solvents, chemical compounds, biological molecules.

The particles of DBM (or inventive DBM material) may also be formed intovarious shapes and configurations. The particles can be formed intorods, strings, sheets, weaves, solids, cones, discs, fibers, wedges etc.In certain embodiments, the shape and size of the particles in the DBMcomposition affect the time course of osteoinductivity. For example, ina cone or wedge shape, the tapered end will result in osteoinductivityshortly after implantation of the DBM composition, whereas the thickerend will lead to osteoinductivity later in the healing process (e.g.,hours to days to weeks later). In certain embodiments, the particle havea length of greater than 2 mm, greater than 1.5 mm, greater than 1 mm,preferably greater than 500 microns, and most preferably greater than200 microns across its widest dimension. Also, larger particle size willhave induce bone formation over a longer time course than smallerparticles. Particles of different characteristics (e.g., composition,size, shape) may be used in the formation of these different shapes andconfigurations. For example, in a sheet of DBM a layer of long half-lifeparticles may be alternated between layers of shorter half-lifeparticles (See U.S. Pat. No. 5,899,939, incorporated herein byreference). In a weave, strands composed of short half-life particlesmay be woven together with strands of longer half-lives.

In one preferred embodiment of the invention, fibrous DBM is shaped intoa matrix form as described in U.S. Pat. No. 5,507,813, incorporatedherein by reference, and Examples 13 & 14 (embedded matrix fabrication)below. The shaped DBM is then embedded within a diffusion barrier typematrix, such that a portion of the matrix is left exposed free of thematrix material. Particularly preferred blocking matrices are starch,phosphatidyl choline, tyrosine polycarbonates, tyrosine polyarylates,polylactides, polygalactides, or other resorbable polymers orcopolymers. Devices prepared in this way from these matrices have acombination of immediate and longer lasting osteoinductive propertiesand are particularly useful in promoting bone mass formation in humanposterolateral spine fusion indications.

In another embodiment of the invention, inventive DBM compositionshaving a pre-selected three-dimensional shape are prepared by repeatedapplication of individual layers of DBM, for example by 3-D printing asdescribed by Cima et al. U.S. Pat. Nos. 5,490,962; and 5,518,680, eachof which is incorporated herein by reference; and Sachs et al. U.S. Pat.No. 5,807,437, incorporated herein by reference. Different layers maycomprise individual stabilized DBM preparations, or alternatively maycomprise DBM layers treated with stabilizing agents after deposition ofmultiple layers.

In the process of preparing improved inventive DBM materials, thematerials may be produced entirely aseptically or be sterilized toeliminate any infectious agents such as HIV, hepatitis B, or hepatitisC. The sterilization may be accomplished using antibiotics, irradiation,chemical sterilization (e.g., ethylene oxide), or thermal sterilization.Other methods known in the art of preparing DBM such as defatting,sonication, and lyophilization may also be used in preparing theimproved DBM. Since the biological activity of demineralized bone isknown to be detrimentally affected by most terminal sterilizationprocesses, care must be taken when sterilizing the inventivecompositions. In preferred embodiments, the DBM compositions describedherein will be prepared aseptically or sterilized as described inExample 11.

Applications

Improved osteogenic compositions of the present invention may be used topromote the healing of bone injuries. The compositions may be used inany bone of the body on any type of injury. The improved DBM compositionhas been designed to produce bone in human patients with similar timingand at a level similar to 10 .mu.g to 100 .mu.g, preferably 200 .mu.g to1 mg of rhBMP on a collagen sponge. For example, specific bones that canbe repaired using the inventive material include the ethmoid, frontal,nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic,incus, stapes, malleus, cervical vertebrae, thoracic vertebrae, lumbarvertebrae, sacrum, sternum, ribs, clavicle, scapula, humerus, ulna,radius, carpal bones, metacarpal bones, phalanges, ileum, ischium,pubis, pelvis, femur, patella, tibia, fibula, calcaneus, talus, andmetatarsal bones. The type of injury amenable to treatment with theimproved DBM include bone defects resulting from injury, brought aboutduring the course of surgery, infection, malignancy, or developmentalmalformation. The inventive material may be useful in orthopaedic,neurosurgical, cosmetic, and oral and maxillofacial surgical proceduressuch as the repair of simple and compound fractures and non-unions,external and internal fixations, joint reconstructions such asarthrodesis, general arthroplasty, cup arthroplasty of the hip, femoraland humeral head replacement, femoral head surface replacement and totaljoint replacement, repairs of the vertebral column including spinalfusion and internal fixation, tumor surgery (e.g., deficit filling),discectomy, laminectomy, excision of spinal cord tumors, anteriorcervical and thoracic operations, repair of spinal injuries, scoliosis,lordosis and kyphosis treatments, intermaxillary fixation of fractures,mentoplasty, temporomandibular joint replacement, alveolar ridgeaugmentation and reconstruction, inlay bone grafts, implant placementand revision, sinus lifts, etc.

Inventive DBM compositions may also be used as drug delivery devices. Incertain preferred embodiments, association with the inventive DBMcomposition increases the half-life of the relevant biologically activeagent(s). Particularly preferred inventive drug delivery devices areused to deliver osteoinductive growth factors. Other preferred agents tobe delivered include factors or agents that promote wound healing.However, inventive compositions may alternatively or additionally beused to deliver other pharmaceutical agents including antibiotics,anti-neoplastic agents, growth factors, hematopoietic factors,nutrients, etc. Bioactive agents that can be delivered using theinventive DBM composition include non-collagenous proteins such asosteopontin, osteonectin, bone sialo proteins, fibronectin, laminin,fibrinogen, vitronectin, trombospondin, proteoglycans, decorin,proteoglycans, beta-glycan, biglycan, aggrecan, veriscan, tenascin,matrix gla protein hyaluronan; cells; amino acids; peptides; inorganicelements; inorganic compounds; organometallic compounds; cofactors forprotein synthesis; cofactors for enzymes; vitamins; hormones; solubleand insoluble components of the immune system; soluble and insolublereceptors including truncated forms; soluble, insoluble, and cellsurface bound ligands including truncated forms; chemokines,interleukins; antigens; bioactive compounds that are endocytosed; tissueor tissue fragments; endocrine tissue; enzymes such as collagenase,peptidases, oxidases, etc.; polymeric cell scaffolds with parenchymalcells; angiogenic drugs, polymeric carriers containing bioactive agents;encapsulated bioactive agents; bioactive agents in time-release form;collagen lattices; antigenic agents; cytoskeletal agents; cartilagefragments; living cells such as chondrocytes, osteoblasts, osteoclasts,fibroblasts, bone marrow cells, mesenchymal stem cells, etc.; tissuetransplants; bioadhesives; bone morphogenic proteins (BMPs),transforming growth factor (TGF-beta), insulin-like growth factor(IGF-1, IGF-2), platelet derived growth factor (PDGF); fibroblast growthfactors (FGF), vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF), growth factor binding proteins, e.g., insulin-likegrowth factor binding protein (IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5,IGFBP-6); angiogenic agents; bone promoters; cytokines; interleukins;genetic material; genes encoding bone promoting action; cells containinggenes encoding bone promoting action; cells genetically altered by thehand of man; externally expanded autograft or xenograft cells; growthhormones such as somatotropin; bone digestors; antitumor agents;fibronectin; cellular attractants and attachment agents;immunosuppressants; bone resorption inhibitors and stimulators;mitogenic factors; bioactive factors that inhibit and stimulate secondmessenger molecules; cell adhesion molecules, e.g., cell-matrix andcell-cell adhesion molecules; secondary messengers; monoclonalantibodies specific to cell surface determinants on mesenchymal stemcells; portions of monoclonal antibodies specific to cell surfacedeterminants on mesenchymal stem cells; clotting factors;polynucleotides; and combinations thereof. The amount of the bioactiveagent included with the DBM composition can vary widely and will dependon such factors as the agent being delivered, the site ofadministration, the patient's physiological condition, etc. The optimumlevels being determined in a specific case based upon the intended useof the implant.

For example, inventive DBM compositions may be prepared so that theyinclude one or more compounds selected from the group consisting ofdrugs that act at synaptic and neuroeffector junctional sites (e.g.,acetylcholine, methacholine, pilocarpine, atropine, scopolamine,physostigmine, succinylcholine, epinephrine, norepinephrine, dopamine,dobutamine, isoproterenol, albuterol, propranolol, serotonin); drugsthat act on the central nervous system (e.g., clonazepam, diazepam,lorazepam, benzocaine, bupivacaine, lidocaine, tetracaine, ropivacaine,amitriptyline, fluoxetine, paroxetine, valproic acid, carbamazepine,bromocriptine, morphine, fentanyl, naltrexone, naloxone,); drugs thatmodulate inflammatory responses (e.g., aspirin, indomethacin, ibuprofen,naproxen, steroids, cromolyn sodium, theophylline); drugs that affectrenal and/or cardiovascular function (e.g., furosemide, thiazide,amiloride, spironolactone, captopril, enalapril, lisinopril, diltiazem,nifedipine, verapamil, digoxin, isordil, dobutamine, lidocaine,quinidine, adenosine, digitalis, mevastatin, lovastatin, simvastatin,mevalonate); drugs that affect gastrointestinal function (e.g.,omeprazole, sucralfate); antibiotics (e.g., tetracycline, clindamycin,amphotericin B, quinine, methicillin, vancomycin, penicillin G,amoxicillin, gentamicin, erythromycin, ciprofloxacin, doxycycline,acyclovir, zidovudine (AZT), ddC, ddI, ribavirin, cefaclor, cephalexin,streptomycin, gentamicin, tobramycin, chloramphenicol, isoniazid,fluconazole, amantadine, interferon,); anti-cancer agents (e.g.,cyclophosphamide, methotrexate, fluorouracil, cytarabine,mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin,mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin,decarbazine); immunomodulatory agents (e.g., interleukins, interferons,GM-CSF, TNF.alpha., TNF.beta., cyclosporine, FK506, azathioprine,steroids); drugs acting on the blood and/or the blood-forming organs(e.g., interleukins, G-CSF, GM-CSF, erythropoietin, vitamins, iron,copper, vitamin B.sub.12, folic acid, heparin, warfarin, coumarin);hormones (e.g., growth hormone (GH), prolactin, luteinizing hormone,TSH, ACTH, insulin, FSH, CG, somatostatin, estrogens, androgens,progesterone, gonadotropin-releasing hormone (GnRH), thyroxine,triiodothyronine); hormone antagonists; agents affecting calcificationand bone turnover (e.g., calcium, phosphate, parathyroid hormone (PTH),vitamin D, bisphosphonates, calcitonin, fluoride), vitamins (e.g.,riboflavin, nicotinic acid, pyridoxine, pantothenic acid, biotin,choline, inositol, camitine, vitamin C, vitamin A, vitamin E, vitaminK), gene therapy agents (e.g., viral vectors, nucleic-acid-bearingliposomes, DNA-protein conjugates, anti-sense agents); or other agentssuch as targeting agents etc.

In certain embodiments, the agent to be delivered is adsorbed to orotherwise associated with the matrix being implanted. The agent may beassociated with the matrix of the DBM composition through specific ornon-specific interactions; or covalent or non-covalent interactions.Examples of specific interactions include those between a ligand and areceptor, a epitope and an antibody, etc. Examples of non-specificinteractions include hydrophobic interactions, electrostaticinteractions, magnetic interactions, dipole interactions, van der Waalsinteractions, hydrogen bonding, etc. In certain embodiments, the agentis attached to the matrix using a linker so that the agent is free toassociate with its receptor or site of action in vivo. In certainpreferred embodiments, the agent to be delivered may be attached to achemical compound such as a peptide that is recognized by the matrix ofthe DBM composition. In another embodiment, the agent to be delivered isattached to an antibody, or fragment thereof, that recognizes an epitopefound within the matrix of the DBM composition. In a particularlypreferred embodiment, the agent is a BMP, TGF-.beta., IGF, parathyroidhormone (PTH), growth factors, or angiogenic factors. In certainembodiments at least two bioactive agents are attached to the DBMcomposition. In other embodiments at least three bioactive agents areattached to the DBM composition.

The growth factor stabilizing strategies described herein, may also beapplied directly to growth factors associated with synthetic matricessuch as ceramics, bone cements, or polymers. In these embodiments one,two, or more growth factors are associated with the synthetic matrix. Agrowth factor is associated with an anchoring matrix (e.g., amorphous orcrystalline calcium phosphate associated with a growth factor such asBMP), wherein the composition is prepared in the presence of a diffusionbarrier such as amylose, fatty acid, or a resorbable polymer, or with acombination of at least two or more of these stabilizing agents. In apreferred embodiment, a poorly crystalline calcium phosphate isassociated with a growth factor mixed with a starch/lecithin diffusionbarrier.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Preparing Demineralized Bone Matrix (DBM)

DBM may be prepared using any method or technique known in the art (seeRussell et al. Orthopedics 22(5):524-531, May 1999; incorporated hereinby reference). The following is an exemplary procedure for preparingdemineralized bone derived from Glowacki et al. “Demineralized BoneImplants” Clinics in Plastic Surgery 12(2):233-241, April 1985, which isincorporated herein by reference. Bones or bone fragments from donorsare cleaned to remove any adherent periosteum, muscle, connectivetissue, tendons, ligaments, and cartilage. Cancellous bone may beseparated from dense cortical bone and processed as large pieces.Cortical bone may be cut into small pieces to improve the efficiency ofsubsequent washes and extractions. Denser bone from larger animals mayneed to be frozen and hammered in order to produce chips less than 1 cm.The resulting pieces of bone are thoroughly washed with cold, deionizedwater to remove marrow and soft tissue.

The cleaned bone is then extracted with frequent changes of absoluteethanol for at least 1 hour. Typically, a total of 4 liters of ethanolis used per 100 g of bone. The bone is then extracted with frequentchanges of anhydrous diethyl ether in a fume hood for 1 hour. Typically,2 liters of ether is used per 100 g of bone. The bone is dehydrated bythese extractions of ethanol and ether and can be stored at roomtemperature.

The dehydrated bone is then frozen and then pulverized in a liquidnitrogen impacting mill. Pulverized bone is then sieved into fractionsof 75 to 250, 250 to 450, and greater than 450 microns. Bone particlefractions are then demineralized using 0.5 M hydrochloric acid (50 mlper gram) for 3 hours at room temperature or at 4 .degree. C. onmagnetic stirrers with insulation to prevent overheating. Large chips ofbone and blocks are extracted completely at 4 .degree. C. with frequentchanges of 0.5 M hydrochloric acid. The demineralization process can bemonitored radiographically, by ashing, or by nondecalcified histologictechniques (von Kossa stain). The acid and liberated minerals are washedaway with cold, deionized water until the pH of the wash matches the pHof the water. The water washes can be decanted from the large particlesand chips of bone; however, the washes must be removed by centrifugationfrom the finer particles. The washing step requires approximately 500 mlof water per gram of starting bone particles.

Demineralized bone powders are extracted with changes of absoluteethanol for 1 hour using 200 ml of ethanol per gram of starting boneparticles. The material is extracted in a fume hood with changes ofanhydrous ethyl ether for 1 hour with 100 ml of ether per gram ofstarting bone particles. After the last change of ether is removed, thedemineralized bone powder is left overnight in the hood until all theresidual ether has vaporized. The particles should be odorless,snow-white, and discrete. To sterilize the demineralized bone material,it may be treated with cold ethylene oxide or irradiated.

To test the bioactivity of the prepared DBM, 25 mg of the material isimplanted into each of two thoracic subcutaneous pockets in shaved,anesthetized 28-day old male Charles River CD rats. The implantedspecimens may then be harvested and inspected several days afterimplantation. The composition of the induced tissue can be quantified byhistomorphometric analysis and be biochemical techniques.

Example 2 Another Method of Preparing DBM

DBM may be prepared using any method or techniques known in the art (SeeRussell et al. Orthopedics 22(5):524-531, May 1999; incorporated hereinby reference).

Demineralized bone matrix was prepared from long bones. The diaphysealregion was cleaned of any adhering soft tissue and then ground in amill. Ground material was sieved to yield a powder with particlesapproximately 100 .mu.m to 500 .mu.m in diameter. The particulate bonewas demineralized to less than about 1% (by weight) residual calciumusing a solution of Triton X-100 (Sigma Chemical Company, St Louis, Mo.)and 0.6N HCl at room temperature followed by a solution of fresh 0.6NHCl. The powder material was rinsed with deionized water until the pHwas greater than 4.0. It then was soaked in 70% ethanol and freeze-driedto less than 5% residual moisture.

Example 3 Formulating Preferred Inventive DBM Compositions

The carrier was prepared by mixing approximately 6.5% (w/w) of themodified starch, B980, with approximately 30% (w/w) maltodextrin (M180)and approximately 63.5% (w/w) sterile, deionized water. The mixture washeated to 70 .degree. C. to pre-gelatinize. The pre-gelatinized mixturewas then transferred into a steam autoclave and sterilized/gelatinizedat 124 .degree. C. for 2 hours. The resulting mixture then had aconsistency of pudding. The cooled carrier mixture was then combinedwith DBM (from Example 2) and water, in a ratio of approximately27:14:9, respectively. The stabilized DBM was then implanted intoathymic rats to assess osteoinductivity.

Alternative embodiments: Other components such as glycerol were added asa solution (approximately 20% w/v) in water instead of water at the timeof pre-gelatinization or during the final composition mixing and werefound to have acceptable handling characteristics.

Example 4 Stabilized DBM

The table below describes the preparation of a variety of inventive DBMcompositions with different stabilizers. All preparations are preparedaseptically, and all preparations may be used with DBM particles,fibers, or solid formed matrices.

1 Class of Stabilizer Stabilizer DBM form Method Diffusion BarrierResorbable polymers in Approximately 150-1000 Pre-swollen particles arepowdered form: micron particles lyophilized and mixed with polymerTyrosine poly arylate then pre-swollen with 100% powder. The mixture isTyrosine polycarbonate glycerol, excess removed by Melt cast at 60-115.degree. C. polyorthoester filtration Following cooling the polymer DBMmonolith is pulverized Phosphatidyl choline Approximately 150-1000 Oneor more of the Phosphatidyl-ethanolami-ne micron particles lyophillizedindicated lipids are blended Squalene with the DBM to prepare a Starchphosphatidyl choline paste containing about 30-80% DBM Masking AgentSuspend in standard buffer Mix lyophilized DBM with Apply DBM particlesas Lectins system for the specific protein protein solution to prepare athick usual or mix with standard Antibodies or PBS, or 1 mMHCl to aslurry (.about.0.33 gm/mL). Re-DBM carrier (e.g., HumanAnti-concentration ranging from lyophilize. glycerol, starch, pluronic)noggin about 1 ng/ml to about 10 prior to application. Human Anti-mg/mlBMP Factor binding proteins Noggin Chordin TGBP Enzyme Inhibitors TIMPsSoybean trypsin Competitive Substrates Poly-lys-arg Di-mannosePoly-mannose Poly-L-lysine

Example 5 In Vitro Assessment of Protective Agents

Samples of DBM with carrier with, and without stabilizing agents (orvarious concentrations and/or formulations of stabilizing agents) areprepared and incubated with serum or individual enzymes (e.g., papain)in pH 7.4 PBS buffer and incubated at 37 .degree. C. for 0.5, 1, 2, 4,8, and 24 hours Samples are then extracted to determine theconcentrations of growth factors and other matrix proteins as outlinedin Ueland et. al. (“Increased cortical bone content of insulin-likegrowth factors in acromegalic patients” J Clin Endocrinol Metab 1999January; 84(1):123-7; incorporated herein by reference). Samples areprepared for native and denaturing SDS gel electrophoresis followed byWestern blot analysis or Western Ligand blotting as described in Uelandet al. (1999) and incorporated herein by reference (Ueland et al“Increased cortical bone content of insulin-like growth factors inacromegalic patients” J Clin Endocrinol Metab 1999 January; 84(1):123-7;and Walker, J. M. (Ed) The Protein Protocols Handbook, Second Edition2002, Humana Press Totowa, N.J.; each of which is incorporated herein byreference).

Samples containing stabilizing agents demonstrating less degradation ofgrowth factors or other proteins than samples without stabilizing agentsare then tested for osteoinductivity at 7, 14, 21, and 28 days in theathymic rat assay. Extract samples can also be tested rapidly forbiological activity in a tissue culture assay as described in Zhang etal. (1997).

Example 6 Determining Time Course for Induction of Bone Growth byIntermuscular Implant

This Example characterizes the time course of induction of bone growthin an intermuscular site using the inventive materials, as compared withDBM base powder (as in Example 1), at time points of 7, 14, 28, and 35days. This Example is similar to the rat model for assessingosteoinduction of DBM found in Edwards et al “Osteoinduction of HumanDemineralized Bone: Characterization in a Rat Model” ClinicalOrthopaedics 357:219-228, December 1998; incorporated herein byreference.

The study was conducted in athymic (nude) rats in order to minimize thepotential for a cross-species incompatibility response to human tissueimplants. The hind-limb intermuscular site was used for the initialdetermination of heterotopic bone induction properties because the sitedoes not naturally contain bone.

Female homozygous mu/mu rats in the 50-75 g range were obtained fromHarlan (Indianapolis, Ind.). The rats were housed for one week foracclimatization purposes prior to surgery. Sterile microisolator cageswere used throughout the investigation, with sterile water and rodentdiet provided ad libitum.

Implant Placement: A single intermuscular (IM) site was utilized in eachhind limb of 30 rats. To provide a common positive control over allanimals, a single 40 mg sample of rat DBM powder was placedintramuscularly within the left pectoralis (LP) muscle of each rat.Animals were allowed normal activities following surgical procedures.

Implant Materials: DBM and test materials were kept at room temperature.Eight 145 mg samples of Test and eight 40-mg samples of DBM powder weretested for implantation times of 7, 14, and 28 days. Six samples of eachwere tested at 35 days. The 40 mg samples of DBM powder were rehydratedwith 100 .mu.l of sterile ALLOPREP™ (Ostetotech, Eatontown, N.J.). Eachof the samples was packed into a 1 ml blunt cut syringe. Implantationwas randomized so that a single animal did not receive two of the sameimplants.

Anesthesia: The rats were anesthetized with a mixture of ketamine (200mg), xylazine (400 mg), and physiological saline (10 ml). The dosage was3.5 ml/kg body weight administered intraperitoneally.

Procedure: Aseptic surgical procedures were carried out in a laminarairflow hood. A 1-cm skin incision was made on each upper hind limbusing a lateral approach, and the skin was separated from the muscle byblunt dissection. A superficial incision aligned with the muscle planewas made to allow for insertion of the tips of the scissors. Bluntdissection was performed from this line deep into the muscle to create apocket to hold the implanted material. A single suture was inserted toclose the muscle pocket, and the skin was closed with metal clips.

Implantation of specimens in the left pectoralis muscles involved makinga 1-cm skin incision over the chest, blunt dissection of the muscle tocreate a pocket, and positioning of the rat DBM powder using a bluntsyringe. A single suture was inserted to close the muscle pocket, andthe skin is closed with metal clips.

Rats were euthanized with CO.sub.2 following the designated implantationtime. Implant materials were located by palpitation, retrieved by bluntdissection, and cleaned of the surrounding tissue by careful trimming.An observer blinded to implant type performed a macroscopic evaluationof the implant material. Color, vascularity, hardness, and integritywere scored according to the scheme outlined in the Table below. (Thehighest score for the most robust response would be a 4 while a specimenshowing little or no osteoinductive potential would score a 0.)Experience with this model has shown a high correlation between visualobservations and histological observations of implant performance onlyat the extremes of both ends of the scale.

2 Macroscopic Observation Scoring Guidelines Color: .sup. White (W).sup..sup. Grey (G) Red (R) Vascularity: None (N) Some (S) sup. Robust(R).sup. Hardness: Mushy (M).sup. .sup. Firm (F) Hard (H).sup.Integrity: Diffuse (D).sup. Flat (F) Nodule (N) Score: 0 0.5 1

Histology: Retrieved materials were fixed in Neutral buffered formalin.After fixation in formalin, samples were decalcified in 10% formic acid,dehydrated in graded alcohols, embedded in JB-4 (glycol methacrylate,Polysciences, Inc., Warrington, Pa.) and sectioned, Five-micron sectionswere stained with toluidine blue and evaluated by light microscopy.

The explants were histologically evaluated using a semiquantitativemethod. Briefly, a numerical score based on a five-point scale wasassigned to each section of nodule: 4=more then 75% involved in new boneformation; 3=51-75% involved in new bone formation; 2=26-50% involved innew bone formation; 1=1-25% of the explant involved in new boneformation; and 0=no evidence for the process of endochondral boneformation including the presence of cartilage or chondrocytes, activeosteoblasts, osteoid, newly formed and mineralized bone, and/or marrowand associated fat cells.

3 Scoring of Histological Sections Score New Bone Formation 0 No newbone formation 1<25% new bone formation 2 26-50% new bone formation 351-75% new bone formation 4>75% new bone formation

Following histological analysis, average scores were calculated for eachmaterial type. Based on pervious experience with this animal model, eachgroup was assigned an assessment of osteoinductive potential based onthe average histological score.

Results: This protocol was followed with the test material, a DBM with astarch stabilizer as described in example 3, as compared with controlGPS1-2 base DBM powder. At the 7-day timepoint, DBM with a starchstabilizer and GPS1-2 powder achieved the same level of induction, witha histologic score of 0.9.+−.0.4 and 1.0.+−.0, respectively. All sampleswere hypercellular with a few chondrocytes present. At the 14-daytimepoint, the DBM with a starch stabilizer achieved a greater level ofinduction than the GPS1-2 powder, with a histologic score of 3.6.+−.0.5and 2.9.+−.1.0 respectively. Clusters of chondrocytes were present inall of the DBM with a starch stabilizer samples. At this time point,half of the powder samples also had clusters of chondrocytes, orscattered cells. At the 28-day point, few chondrocytes were present ineither the DBM with a starch stabilizer or the GPS1-2 powder. Mostsamples exhibited mature bone by this stage. Some tissue infiltrationwas noted in three of the DBM with a starch stabilizer samples and twoof the powder samples. The histologic score for the DBM with a starchstabilizer samples and two of the powder samples. The histologic scorefor the DBM with a starch stabilizer remained constant after the 14days, whereas the histologic score for the powder improved from2.9.+−.1.0 to 3.9.+−.0.4 between days 14 and 35 days, withoutsignificant change noted for those samples at the 35-day time point

4 Mean Histologic Scores Product 7-Day 14-Day 28-Day 35-Day DBM (GPS1-2)with a 0.9.+−.0.4 3.6.+−.0.5 3.6.+−.0.5 3.5.+−.0.8 starch stabilizer DBM(GPS1-2) powder 1.0.+−.0 2.9.+−.1.0 3.9.+−.0.4 3.7.+−.0.5 (Control)

Conclusions: The results of this study indicated that the rate ofinduction for the DBM with a starch stabilizer increased to the 14-daytimepoint and remained elevated through the end of the time course. TheGPS1-2 powder exhibited induction at a slower rate at the 14-day timepoint but was the same as the DBM with a starch stabilizer samples by 28days. At this point, the osteoinductive potential for both products wasnearly the same with only a difference of 0.3 in mean histologic scoresand remained the same at the 35-day time point. The DBM with a starchstabilizer sample showed a faster rate of bone formation compared to thepowder control. The qualitative evaluation of increased number ofchondrocytes present was indicative of increased bone formation in theDBM with a starch stabilizer samples.

Example 7 Evaluating Efficacy of Inventive Compositions in Healing BoneDefects

Background Information: Morselized autogenous cancellous bone (ABG) haslong been considered the “gold standard” for osteoinduction when a bonegraft is required in an orthopedic clinical situation. Unfortunately,the amount of ABG available is limited, and there is at least a 5%surgical morbidity associated with the harvesting procedure.Demineralized bone matrix (DBM) has been shown to have equal to superiorhealing potential to ABG. One of the major disadvantages todemineralized bone matrix is that it often does not hold the threedimensional space of the defect. Thus, invasion of the defect siteoccurs from the surrounding muscle tissue. The test article, DBM with astarch stabilizer, offers a semi-solid texture so that the threedimensional space was maintained.

The rabbit ulna defect model has been modified and used in numerousprojects to test the efficacy of osteoinductive and osteoconductivegrowth factors and matrices as substitute to autogenous bone graft. Theaim of this study was to evaluate the bone inducing capacity of the newDBM formulation grafting material in comparison to previous formulationsand ABG.

Materials and Methods:

Study Design Summary:

A. Rabbit bilateral 2-cm ulnar defects.

Treatment groups:

1. DBM+starch

2. Starch Carrier alone

3. Autograft (historical data used for comparison)

Surgical Procedure: Six months old male New Zealand white rabbits wereused. A 2.0 centimeter non-uniting defect was surgically created in thebilateral ulnae of all rabbits. After complete periostectomy, thoroughdefect wash, and partial diaphyseal wash, grafting was implanted(according to test groups) via open surgical technique into each defect.The wound was closed primarily in layers. The test groups are listed inthe table below. When anesthesia was achieved, both forelimbs wereshaved and prepared with the rabbit supine (limbs up) position.Longitudinal incisions (3-4 cm) were made over both ulnae and thediaphysis (midshaft) portion of the ulna was exposed. The distalosteotomy was made 1 cm from the ulnocarpal (wrist) joint and theproximal osteotomy made 3.0 cm from the ulnocarpal joint, to create a 2cm defect. The osteotomies were created with a high speed burr. Theresultant loose block of diaphyseal bone was excised with its periosteumintact. Due to the very adherent interosseous membrane of the rabbitforelimb, internal fixation was not required. After irrigation withsterile saline to remove blood, bone, and marrow remnants, the implantmaterial was placed in the defect. The deep fascial layer was closed asan envelope around the defect with 3-0 chromic suture. The skin wasclosed with interrupted nylon suture. A post-operative dressing/splintwas applied and removed on the fourth post-operative day.

Radiographs: Antero-posterior radiographs were obtained immediatelypost-operatively and additional radiographs were taken at 3, 6, 9, and12 weeks. High resolution (Faxitron) radiographs were taken of bothlimbs after excision and cleaned of soft tissue at either 6 or 12 weeks.Three blinded observers assess each time point for bone formation andremodeling.

Results: In vivo radiographs at 3 weeks indicated bone formation wasevident in the starch-based formulation (FIG. 1). At 6 weeks,trabeculation was observed and almost complete bridging of thecritical-sized defect with the starch-based formulation (FIG. 2).

Conclusion: The starch-based formulation appeared to improve the rate atwhich bone formation developed.

Example 8 The Following Table Summarizes the Results of Biocompatibilityand Safety Studies for the Starch-Based Diffusion Barrier DBMFormulation of Example 3

All studies listed in the table below, with the exception of study #12,were performed by NAMSA (North American Science Associates, Inc.)—ISO9001 certified and fully accredited by the Association for Assessmentand Accreditation of Laboratory Animal Care International (AAALAC). FDAguidelines were followed and NAMSA is registered with the USDA. Allsamples submitted to NAMSA were tested according to laboratory qualityguidelines necessary to assure valid data.

5 Starch Diffusion Barrier DBM Formulation Testing MATERIAL ABSORBEDWITHIN 30 DAYS PASS/FAIL—No. TEST NAME TEST MATERIAL MODEL METHODCOMMENTS 1 Cytotoxicity Saline Extraction—In vitro assay—48 hour readPass—Non-Carrier L-929 mouse cytotoxic (M180/B980) fibroblasts (4 g:20ml extract) 2 Hemolysis Saline Extraction—In vitro assay—Pass—NonCarrier rabbit blood hemolytic (M180/B980) (4 g:20 ml extract) 3 PyrogenSaline Extraction—Intravenous Repeated Pass—Non-Carrier injection—Rabbitmeasures 1-3 pyrogenic (M180/B980) hours post (4 g:20 ml extract)injection 4 Genotoxicity a. Saline Extraction—In vitro—Ames Measurementa. Pass—Non-Carrier (M180/B980) Assay of revertants mutagenic (4 g:20 mlextract) b. Pass—Non b. DMSO Extraction—mutagenic Carrier (M180/B980) (4g:20 ml extract) 5 Acute a. Saline Extraction-Saline extract—4, 24, 48,72 a. Pass—Non Systemic Carrier (M180/B980) IV injection—hour readstoxic Toxicity (4 g:20 ml extract) mice b. Pass—Non b. Cottonseed oilCottonseed oil toxic Extraction-Carrier extraction —IP (M180/B980)injection—mice (4 g:20 ml extract) 6 Sensitization a. SalineExtraction-Maximization Induction I Induction I: Carrier (M180/B980)Assay—Guinea (zero time) no (4 g:20 ml extract) pigs Induction II (6abnormalities b. Cottonseed oil days) detected Extraction-Challenge (13Induction I: Carrier (M180/B980) days post no (4 g:20 ml extract)induction II)—abnormalities Topical Application 24, 48, 72 hour detectedreads Challenge: No evidence of causing delayed dermal contactsensitization 7 Intracutaneous a. Saline Extraction-0.2 ml subcutaneous24, 48, 72 hour a. Pass—Non Reactivity Carrier (M180/B980) injection @five reads irritant (4 g:20 ml extract) separate sites of b. Pass—Non-b.Cottonseed oil each of 3 rabbits irritant Extraction-Carrier (M180/B980)(4 g:20 ml extract) 8 Muscle a. Final product 6 .times. 2 ml portions 1,2, 4 and 24 Complies with Implantation (rabbit specific) overdorsolumbar hour reads clearance of <Study (Pilot) b. Carrier(M180/B980) region 30 days Clearance in <1 week 9 Muscle a. Finalproduct Rabbit, Surgical 3 and 7 days a. Pass—non-Implantation (rabbitspecific) Method reads irritant Study—Histo-b. Carrier (M180/B980) 0.2ml implanted Irritation and b. Pass—non-pathology c. Rabbit DBM toxicityirritant powder alone evaluation a. Pass—non-irritant 10Implantation—Final Dog; 3 time points: Clearance<2 ClearanceFormulation—Intramuscular 2, 4, 6 weeks. weeks Study Dog specificImplantation To Subsequent Absorption; Site reads subject Adjacent toRibs; to 30 cc volume. bioresorption N=3 profile 11 Systemic a. Finalproduct Rabbit Sacrifice time Pass Toxicity—(rabbit N=4 for each pointsat 7, 14, Histopathology—Intramuscular specific) dose@ each 28, 60 and90 No evidence Implantation b. Rabbit DBM time point days subject to ofsystemic powder alone N=3 DBM absorption toxicity Using Direct powderprofile. Blood No evidence of Contact controls@ and urine at 72 carrierat 7 Implantation each time point hours. days Low (high clinicalSurgical Blood and Termination of dose, 1.times. @ implantation urineanalysis study at 60 3.8 g/rabbit @ along vertebral with histologydays—No approx 1 g/kg) and column and (liver and changes in High dose(5.times. along ribs for kidney); hematology or high clinical dose) highdose Evaluation for clinical implantation gross chemistry anatomicalvalues; lesions No evidence of systemic toxicity; Evidence of ectopicbone formation 12 Femoral Final Formulation Rat 12 Weeks; RadiographicDefect Histology and evidence of X-rays bone formation

Biocompatibility of DBM with a starch stabilizer. Clearance studiesconfirmed the removal of DBM with a starch stabilizer carrier from theimplant site in less than 30 days, classifying it as a class Btissue/bone implant category for ISO 10993 biocompatibility studies.Four evaluation tests for consideration for Class B tissue/bone implantcategory are listed in the ISO guidelines. They are: cytotoxicity,sensitization, implantation and genotoxicity. Acute systemic toxicitymay also apply in specific cases. In addition to the suggested fourtests, a total of nine additional safety, biocompatibility, and efficacystudies were performed (including Example 7). These studies aresummarized in the table above.

Local Reactions

A. Acute intracutaneous injection, and acute muscle implantation studieswere performed. The intracutaneous studies involved both saline andcottonseed oil extracts of a starch stabilizer. DBM with a starchstabilizer prepared with Rabbit DBM was used for muscle implantation.DBM with a starch stabilizer produced minimal irritation in bothstudies, being deemed a non-irritant when compared to the positivecontrol in the muscle implantation and having a primary indexcharacterization of negligible when administered as an intracutaneousextract. Intramuscularly applied DBM without starch carrier, was foundto be a moderate irritant.

B. Cytotoxicity and Genotoxicity. Extracts of a starch stabilizerdemonstrated no ability to induce cell lysis or bacterial mutagenicity.The cell lysis study employed a saline extract of DBM with a starchstabilizer. The genotoxicity studies utilized both saline and DMSOextracts tested on two bacterial species: S. typhimurium and E. coli.

C. Hemolysis and Pyrogenicity. Saline extracts of a starch stabilizerwere deemed to be non-pyrogenic and non-hemolytic. Body temperatures inrabbits injected with saline extracts of DBM with a starch stabilizergave no indication of pyrogenicity, and the extract produced a hemolyticindex of 0 when added to anticoagulated pooled rabbit blood.

D. Sensitization. Extracts of the carrier showed no evidence of delayeddermal contact sensitization. This study employed a saline andcottonseed oil extracts of the carrier. Guinea pigs received anintradermal injection of the extracts and, following a recovery period,were subsequently challenged with a patch of the extract material.

E. DBM with a starch stabilizer Systemic Safety/Tox. No evidence oftoxicity was observed in studies in which DBM with a starch stabilizer(rabbit DBM) was implanted intramuscularly (high dose also hadsubcutaneous, see below) in the paravertebral muscle, and animalsmonitored for 60 days. In these studies, rabbits were implanted witheither approximately 3.5 cc (low dose) or 17.5 cc (high dose) of DBMwith a starch stabilizer (.about.1.1 gm/cc). The doses on a gm perkilogram basis (.about.1.3 gm/kg; .about.6.41 gm/kg) are approximatelyequivalent to 5.6.times. and 28.times. average human implantation dose(15 cc/70 kg or 0.23 gm/kg) respectively. In the case of the high dose,due to space limitations in the paravertebral implant site, only 3.5 ccof DBM with a starch stabilizer were implanted paravertebrally and theremaining 14 cc were implanted subcutaneously in the dorsal thorax.

Necropsy results for the test animals failed to show any treatmenteffect. Blood chemistries and urinalysis values all fell within thenormal range, with the exception of serum alkaline phosphatase which wasexpected to increase due to the induction of ectopic bone formation inresponse to DBM with a starch stabilizer.

Example 9 Assaying Osteoinductivity of Test Materials

Objective: The goal of this Example is to assess the characteristics ofvarious potential Protective Agents, and particularly to identify thosewith no negative impact on osteoinductivity. Preferably, the ProtectiveAgents are easy to handle, irrigatable, non-toxic, degradable, andmoldable (preferred consistency resembles plumber's putty).

Methods and Materials: This study is conducted using an athymic (nude)rat model. Preferably, a single DBM preparation is utilized in allformulations. Potential Protective Agent materials are sterilized byirradiation. Various Test Compositions, and control DBM, are implantedinto animals, 6-8 sites per material. Each animal received bilateralintramuscular implantations into the hindlimbs Each Test Compositioncontains 40 mg DBM per bone site. The volume can vary depending on thenature of the carrier.

Results: This protocol was applied to four different Test Compositions,plus control DBM. The Test Compositions were implanted into 30 animals;DBM was implanted into 8 individual animals. Protective Agents weresterilized by autoclaving. The following Protective Agent Solutions wereprepared:

6 G 8% Starch, 28.5% Maltrin 180: 2.8 g Autoclave for 40 minutesStarch+22.2 g DI water before addition of 10 g Maltrin M180H 8% Starch,12.7% glycerol, 28.5% Maltrin Autoclave for 40 minutes 180: 2.8 gStarch+22.2 g 20% glycerol before addition of 10 g solution Maltrin M180

The following Test Composition formulations were prepared:

7 Implant Preparation Name Composition Recipe E 8% Starch (n=8) 0.4 g ofDBM powder mixed with 0.80 g of solution E. Mix thoroughly. F 8% Starch,18.4% glycerol 0.4 g of DBM powder mixed with (n=8) 0.80 g of solutionF. Mix thoroughly. G 8% Starch, 28.5% Maltrin 0.4 g of DBM powder mixedwith 180 (n=8) 0.73 g of solution G. Mix thoroughly. H 8% Starch, 12.7%glycerol, 0.4 g of DBM powder mixed with 28.5% Maltrin 180 (n=8) 0.73 gof solution H. Mix thoroughly Control Human Powder (HDBM)

In addition, as a positive control in every animal, RDBM was placed inthe left pectoralis.

Results:

8 Implant Material Mean SD HDBM—Control Human Pool 2.9 1.0KF-135-040501-10 Sample E 3.4 0.9 Sample F 3.8 0.5 Sample G 3.6 0.8Sample H 3.3 1.2

Conclusion: No Test Composition had a negative impact onosteoinductivity.

Example 10 Osteoinduction in a Rabbit Model

Introduction and methods: Fifty-five male New Zealand White rabbits wereassigned to three treatment groups. Test article was prepared asdescribed in Example 3. Those animals assigned to the Low Dose treatmentgroup (n=20) received 3.5 ml of the test article in the rightparavertebral muscle following a protocol specified procedure. Animalsassigned to the High Dose treatment group (n=20) received 3.5 ml of thetest article in the right paravertebral muscle and 7.0 ml of the testarticle in the subcutaneous tissue of each side of the dorsal thoracicarea. The animals assigned to the Control treatment group (n=15) wereimplanted with 3.5 ml of control article (rehydrated DBM powder) in theright paravertebral muscle. At 7, 14, and 28 days post-implantation,four animals from the Low and High Dose treatment groups and threeanimals from the Control groups were humanely sacrificed. At 60 dayspost-implantation, the remaining animals were sacrificed (eight from theLow and High Dose test groups and six from the Control treatment group).The implant sites were collected from each rabbit and fixed in 10%neutral buffered formalin (NBF). The test and control implant sites fromthe 60 days post-implantation study interval were placed indecalcification solutions for 3 days after adequate formalin fixation.All tissue samples were processed using standard histologicaltechniques, sectioned at 5 .mu.m, and stained with hematoxylin andeosin.

Results: Osteoinduction was noted in the subcutaneous and intramuscularimplant sites for the test article and in the intramuscular site for thecontrol DBM (no subcutaneous implantation at 28 days post-implantation).New bone was characterized histologically by being slightly moreeosinophilic than the demineralized bone components of the test andcontrol articles. The new trabeculae were lined by plump (active)osteoblasts, osteogenic precursors, osteoid, and poorly mineralizedosteoid. In many cases there were osteocytes present and some evidenceof osteonal remodeling. In some cases cartilage was present. At 60 dayspost-implantation, the new bone was similarly characterized, butassociated with increased thickness, remodeling, and frequently loosefibrovascular stroma (morphologically the same as observed in bonemarrow) containing hematopoietic tissue was observed between thetrabeculae. Subjectively, the test article had a greater degree of boneformation in the muscle implant sites than the control article. Theamount of cartilage present varied between the implant sites. Thisvariation was most likely due to differences in the microenvironment forthose individual implants. The precursor cells involved in new boneformation are pluripotential and under certain microenvironmentalconditions will form fibrous tissue, cartilage, or bone. The cartilagewithin the implant sites undergoes endochondral ossification and becomesbone. Any differences in the tissue response, bone formation, orcartilage formation between the test article implanted within thesubcutaneous tissue and that implanted in muscle were due to anatomicaland microenvironmental differences between the two tissues. Boneformation was noted for both the test and control article implant sites28 days post-implantation. The amount and maturity of the bone (asevident by the amount of remodeling and the presence of loosefibrovascular stroma and hematopoietic tissue) was greatly increased at60 days for the test article.

9 Presence of New Bone and Cartilage by Treatment Group and TimePost-Implantation 7 Days Post-Implantation 14 Days Post-28 Days Post-60Days Post-Treatment Group Bone/Cartilage Implantation ImplantationImplantation High Dose 0/0 (n=4) 0/0 (n=4) 2.0/1.5 (n=4) 3.5/0.0 (n=8)Muscle Subcutaneous 0/0 (n=8) 0/0 (n=8) 1.4/1.5 (n=8) 2.7/0.9 (n=15) LowDose 0/0 (n=4) 0/0 (n=4) 2.3/0.8 (n=4) 4.0/0.4 (n=5) Muscle Control 0/0(n=4) 0/0 (n=4) 0.7/0.7 (n=4) 2.5/0.2 (n=6) Muscle

The ratings in the table above were based on a 0-4 scale with 0 being 0%of implant area occupied by new (viable) bone/cartilage; 1 being 1-25%of implant area occupied by new (viable) bone/cartilage; 2 being 26-50%of implant area occupied by new (viable) bone/cartilage; 3 being 51-75%of implant area occupied by new (viable) bone/cartilage; and 4 being76-100% of implant area occupied by new (viable) bone/cartilage.

Example 11 Terminal Sterilization

This example describes a terminal sterilization method which minimizesosteoinductivity loss in the inventive preparations.

The inventive DBM preparations are produced in a clean room environmentfrom human tissue. The finished implants are placed in individual traypackages.

Each tray is placed in an Audionvac sealing apparatus (Audion Electro B.V., Weesp-Holland) which is supplied with a cylinder consisting of 50/50hydrogen/argon gas. Before the tray packages are sealed, they areevacuated and backfilled with the gas mixture twice. Following sealing,the gas mixture remains in each tray package.

The packaged implants are then sealed packages and then treated with 15KGy gamma radiation from a cobalt 60 source to reduce the bioburden ofthe implants to the desired level.

Example 12 Comparing Osteoinductivity of DBM Preparations to BMP and/orOther Growth Factors

In the series of studies presented here, hybrid recombinant human BMPs(hybrid rhBMPs, hrhBMPs) were prepared possessing strongerheparin-binding epitopes at the N-termini compared with the wild typeBMP. The heparin-binding site enhances binding to the ECM increasinglocal residence time of the BMP so that the potential for interactionwith the appropriate cells in vivo is maximized (Kubler et al. “EHBMP-2.Initial BMP analog with osteoinductive properties” Mund KieferGesichtschir. 3 Suppl 1:S134-9, 1999; incorporated herein by reference).

The aim of the studies was to compare the osteoinductive potential ofhybrid rhBMP-2.times. (hrhBMP-2.times.) with wild-type BMP-2 (rhBMP-2)to determine whether a synergistic potential existed whenhrhBMP-2.times. was combined with a demineralized bone matrix or adevitalized (inactivated bone matrix).

Methods

To assess the osteoinductive activity of the hybrid rhBMP-2.times., 1,5, and 10 .mu.g hrhBMP-2.times. were placed onto 200 mg osteoinductivehuman demineralized bone fiber (DBF) matrix and implantedheterotopically in athymic rats for 21 days (n=6 per group). The DBFmatrix was prepared so that the osteoinductive potential wasapproximately 50% of that usually seen so that differences betweentreated and untreated DBM were evident. Controls consisted ofosteoinductive human DBF matrix alone, inactivated human DBF matrixalone (“devitalized”, GuHCl extracted) and inactivated human DBFcombined with 1, 5, and 10 .mu.g hrhBMP-2.times., active and inactivatedmatrix with 5 .mu.g wild-type BMP-2. All samples were measuredhistologically using a 5-point scoring system (score 4=>75% of thecross-sectional area of the implant with evidence of bone formation,3=51-75%, 2=26-50%, 1=1-25%, 0=no bone formation) (Edwards J T, DiegmannM H, Scarborough N L. Osteoinduction of human demineralized bone:characterization in a rat model. Clin. Orthop. 357:219-28, 1998;incorporated herein by reference).

Results

Histological scoring as described in the methods section was inadequatefor scoring most of the samples that contained a morphogen. Thedevitalized sample alone (inactive DBF matrix) scored 0; devitalized+1.mu.g hrh-BMP-2.times. scored 0.8.+−.0.4; DBF matrix scored 2.5.+−.0.8;all other samples scored 4.

To further distinguish the extent of development of the nodules, aqualitative scoring system was devised to determine the vascularity ofthe sample and residual DBF remaining in the sample. The followingscales were used:

Vascularity (bloody marrow): none=0; minor=1; few vessels, smallvessels=2; moderate cellularity and vessel size=3; extensivecellularity, large vessels=4

Residual DBF: none=0; minor=1; low=2; moderate=3, extensive=4

The active DBF matrix treated with hrhBMP-2.times. produced a moredifferentiated nodule with little residual DBM present, extensive newbone formation and highly developed vasculature which was not evident inthe devitalized group even at the highest concentration of morphogen.The devitalized carrier can be compared to the collagensponge—essentially an inert, 3-dimensional structure to support bonegrowth. The wild-type rhBMP-2 produced a well developed vasculature andmarrow however, the residual bone content was far greater that theactive DBF counterpart.

Conclusion:

The results show that the modified hrhBMP-2.times. possessed strongerosteoinductive properties than its corresponding wild type. Ossificationwas accelerated and the induced bone tissue showed a denser structure.Synergistic results were obtained when hrhBMP-2.times. was combined withactive DBF matrix and not devitalized DBF. The most likely explanationfor these findings is the longer half-life of the hrhBMPs-2.times. atthe implantation site. The persistence of the growth factor at the siteallowed for longer interaction time with local cells rather thanleaching into the surrounding tissues resulting in ectopic boneformation sites. An active matrix substantially increased theosteoinductive properties of the exogenously added growth factorpresumably due to the combined interactions of many growth factorsalready present in demineralized bone (Kubler et al. “Allogenic bone andcartilage morphogenesis. Rat BMP in vivo and in vitro” J.Craniomaxillofac. Surg. 19(7):283-8, 1991; Kubler et al. “Effect ofdifferent factors on the bone forming properties of recombinant BMPs”Mund Kiefer Gesichtschir. 4 Suppl 2:S465-9, 2000; each of which isincorporated herein by reference).

Example 13 Process of Making a Species-Specific Osteoimplant withDefined Dimensions

Long bones from human Rhesus Monkey, canine, and rabbit were used toprepare species-specific solid formed implant matrices. Bones wereaseptically cleaned. The cortical bone was processed in the bone millingapparatus described in U.S. Pat. No. 5,607,269, incorporated herein byreference, to yield about 65 grams of elongate bone fibers. The elongatebone fibers were placed in a reactor and allowed to soak for about 5-10minutes in 0.6 N HCl plus 20-2000 ppm nonionic surfactant solution.Following drainage of the HCl/surfactant, 0.6 N HCl at 15 ml per gram oftotal bone was introduced into the reactor along with the elongate bonefibers. The reaction proceeded for about 40-50 minutes. Followingdrainage through a sieve, the resulting demineralized elongate bonefibers were rinsed three times with sterile, deionized water at 15 mlper gram of total bone, being replaced at 15-minute intervals. Followingdrainage of the water, the bone fibers were covered in alcohol andallowed to soak for at least 30 minutes. The alcohol was then drainedand the bone fibers were rinsed with sterile, deionized water. The bonefibers were then contacted with a mixture of about 4.5 ml glycerol pergram of dry bone fibers and about 10.5 ml sterile deionized water pergram of dry bone fibers s for at least 60 minutes. Excess liquid wasdrained and the resulting liquid composition containing approximately11% (w/v) demineralized, elongate bone fibers was transferred to a 11cm.times.11 cm mold containing a lid having a plurality of protrudingindentations (approximately 1.5 cm.times.3.5 cm in width and length, and4 mm in depth), the lid was gently placed on the mold such that theindentations became immersed into the fibers to exert as little pressureon the composition as possible. The dimensions of the protrusions can bemade specific for the size of the osteoimplant required for the animalmodel of interest. The resulting cut pieces had dimensions of 4.5 cm inlength, 2.5 cm in width and about 8 mm in height (or thickness) withtrough dimensions 3.5 cm in length, 1 cm in width and depth of the of 4mm. The mold was then placed in an oven at 46 .degree. C. for 4 hours.The composition was then frozen overnight at −70 .degree. C. and thenlyophilized for 48 hours. Following lyophilization, the mold wasdisassembled and the sponge-like formed composition was cut intoindividual pieces that contained troughs.

The resulting composition was cohesive, flexible, sponge-like with anobvious continuous three-dimensional structure with visible open pores,had a defined shape including the indentations made by the lidprotrusions, did not require rehydration before use, but was rapidlyhydratable and retained its shape once wetted with fluids and freezingwas not required for storage.

Example 14 Method for Fabricating a Partially Embedded DBM/PolymerComposite

The following method is used to produce a demineralized bone matrixpartially embedded in a resorbable polymer. Such partially embedded DBMsprovide an initial level of osteoinductivity from the non-embedded DBMportion, and then a continuous source of un-degraded active DBM as thepolymer degrades with time. The method is particularly well suited forembedding DBM in tyrosine polycarbonate DT (Integra life sciences) andpoly (L-lactide-co-D, L-lactide 70/30) (Boehringer Ingelheim). Thisdevice has particular application in posterior lateral spine fusion,where it can be placed within the lateral gutter to promoteintertransverse process bone formation. The method can be used to halfembed an appropriately shaped matrix produced by the method described inExample 10 above, or alternatively, a collection of demineralizedcortical bone fibers, where the fibers, are cut approximately 1 inch inlength and arranged in a cylindrical bundle with the long axes of thefibers substantially parallel with one another can be partially embeddedby this method.

A stainless steel adjustable diameter circular clamp, approximately ½inch in height is used to hold the ground polymer, along with the lowerportion of the demineralized bone. The fiber bundle or matrix sample iscentered in the clamp, leaving space around the inside periphery of theclamp to receive the ground polymer material. Heat is then applied tothe underside of the clamp until the polymer has melted. The clamp isthen tightened (diameter reduced) while the polymer is still flowable,forcing the polymer to flow into the lower part of the fiber bundle. Thepolymeric material is then allowed to cool and the clamp removed,embedding the lower portion of the fibers in the solid polymer.

In preferred embodiments resorbable polymers are employed. Temperaturesare used which melt the polymer to a suitable viscosity to allow themelted polymer to flow in and around the demineralized bone. Most oftenthe temperature employed will be from about 0 to about 15 degrees abovethe glass transition temperature of the polymer. Since the biologicalactivity of DBM may degrade if maintained at temperatures above 60.degree. C. for significant periods of times, the preferred polymerswill have glass transition temperatures lower than 100 .degree. C.preferably lower than 80 .degree. C. and most preferable below 60.degree. C. For tyrosine polycarbonate DT a temperature of 115 .degree.C. for 10 minutes is employed. For poly (L-lactide-co-D, L-lactide70/30) 70 .degree. C. is suitable. This method is also applicable if asuitable polymer solvent is used instead of heat to facilitate polymerflow.

Example 15 DBM Preparation Comprising a Mixture of Stabilized DBMs witha Prolonged Half-Life Diffusion Barrier

Two demineralized bone formulations are prepared:

Demineralized bone preparation #1. DBM is prepared from about 150-1000micron bone particles demineralized, lyophilized and then pre-swollenwith 100% glycerol, excess glycerol is removed by filtration. Lactomer9-1, a caprolactone glycolide & calcium stearoyl lactylate (Tyco Inc.North Haven, Conn.) is mixed 10:1 by weight to homogeneity with the DBM.The mixture is melt cast in a mold at 70 .degree. C. Following cooling,the polymer DBM monolith is pulverized in a cryomill and sieved to aparticle size of about 130-1200 microns.

Demineralized bone preparation #2: A lecithin based DBM preparation isprepared according to the method of Han et al “Synergistic Effects ofLecithin and Human DBM on Bone Induction in Nude Rats” Abstract from the28.sup.th Annual Meeting of the Society for Biomaterials (2002)incorporated herein by reference. Briefly, Pospholipon 90G, (AmericanLecithin Company) is mixed with demineralized bone at a weight ration ofbetween 40% lecithin: 60% DBM to 60% lecithin: 40% DBM

A third starch based demineralized bone is prepared according to Example2 with the exception that only one third of the total demineralized bonewas added to the starch carrier. In place of the remaining two thirds ofdemineralized bone, is added equal amounts demineralized bone frompreparations #1 & #2 of this Example. The composition is then mixed toform the implant preparation.

Example 16 Competitive Substrate

Poly-L-lysine may be used as a competitive inhibitor for serine proteaseenzymes. This example describes the preparation of demineralized boneincorporating poly-L-lysine. Poly-L-lysine (10-300 kD) is prepared in 1mM HCL in a range of concentrations from about 1-10 mg/ml. Demineralizedbone is prepared. Following final washing it is mixed with thepoly-L-lysine solution in one of 5 concentrations to form a thick slurry(.about.0.33 gm/mL). The demineralized bone/substrate mixture islyophilized to dryness. The demineralized bone thus prepared is useddirectly or formulated with a carrier.

Example 17 A Fatty Acid/Starch Diffusion Barrier Matrix

Demineralized bone is prepared as described in example 14 with themodification that the polymer/DBM preparation is omitted, being replacedby an equal weight of the lecithin preparation.

Example 18 Osteoinduction of DBM Composition in an Athymic Rat Model

The purpose of this Example is to evaluate the osteoinductive potentialof DBM compositions using a heterotopic osteoinductive 28-day implantmodel (Edwards et al., Clin. Orthop. Rel. Res. 357:219-228, 1998; Urist,Science 150:893-899, 1965; each of which is incorporated by reference).The DBM composition includes cuboidal shaped DBM particles incombination with DBM fibers (See U.S. Ser. No. 60/159,774, filed Oct.15, 1999; WO0232348; each of which is included herein by reference).Chondrocytes are the predominant cell type in the cube of the DBMfollowing 28-day implantation. This study extends the implant time to 49days to look evidence of continued bone remodeling within thedemineralized cortical cube.

Materials and Methods: Equal volumes of crunch samples weighingapproximately 600 mg were packaged in 2.5 ml blunt tipped syringes.Eighteen female athymic rats were obtained from Harlan Sprague DawleyInc. (Indianapolis, Ind.). Animals weights at the time of surgery rangedbetween 186 g and 236 g. 28-day and 49-day implants were evaluated.

The implant sites were assessed histologically. The fiber component wasscored independently of the cubes and was assigned a numerical scorebased on a 5 point semiquantitative scale based on percent of fiber areainvolved in new bone formation. The cube portion was assigned a scorebased on the percent of central Haversian systems involved in new boneformation.

Results: New bone, marrow, and adipocytes were present throughout thefiber portion of the nodules. Chondrocytes were present within thecentral Haversian systems at all time points. At the 28-day time point,the mean osteoinductive score for the fiber portion was 3.1.+−.0.5 forthe fiber portion 89.8.+−.5.8% of the Haversian canals occupied in thecube portion. Cubes were surrounded by new bone or marrow and pockets ofchondrocytes occurred within and between cubes.

The mean osteoinductive score at the 49-day time point was 3.5.+−.0.5for the fiber portion with 98.1.+−.2.4% of the Haversian canals occupiedin the cubes. The notable differences from the 28-day samples includedalmost complete remodeling of the fiber portion, large pockets ofchondrocytes and areas of new bone within the cubes and remodeling atthe edges of the cube.

Conclusions: The cortical cubes play an important role in theosteoinductivity of the DBM composition. The cubes are cut from corticalbone and the central Haversian canals provide a natural porosity.Cartilage persisting after 28 days coincides with a delay in boneformation presumably due to the delayed vascular ingrowth. At 49 days,the cubes showed evidence of remodeling albeit slower than the fibers.Bone remodeling occurred faster on the external surfaces compared tointernal surfaces. The cubes continue to provide the important supportmatrix and osteoinductive signal required for normal bone formationthroughout the healing response.

Example 19 Establishment of Handling Characteristics for InventiveCompositions

The following example describes the addition of demineralized bone to aninventive stabilizing agent and/or diffusion barrier to produce aformable osteoinductive implant composition. The example describes theestablishment of an appropriate carrier viscosity, mixing the carrierwith DBM, and adjustment of the final handling properties of thecompeted composition.

Carrier Viscosity. The inventive starch based compositions were preparedas described in Example 3, with a variety of starch to water ratiosranging from about 5% to about 45%. The starch powders was mixed withwater and the mixture was autoclaved to produce a sterile hydratedstarch preparation. The autoclaved starch was then tested for viscosity.Starch formulations with viscosities within the range of 5000 to 20000sCp were used to prepared DBM compositions.

A Brookfield Viscometer (HB-DV III+) with the appropriate sample adaptor(SSA27/13RPY s/n RP66162 with spindle #27), supported by Rheocalc32software was used to determine the viscosity of the starch carrier.

Mixing of carrier and DBM. The starch carrier with a viscosity ofapproximately 5000 sCpi was mixed with varying quantities of DBM (fromabout 10% to about 50% DBM by weight) to produce a composition with aconsistency similar to that of modeling clay or bread dough. Variationsemploying lesser amounts of DBM resulted in a composition with acohesive yet almost flowable product. Formulations employing more DBMproduced a product with a very stiff consistency, and formulations withhigh levels of DBM became crumbly and fragmented while mixing. Theseformulations were then quantitatively assessed for handling as describedbelow.

Assay for composition handling properties. The following method was usedto establish consistency in handling properties of the inventivecompositions. Compositions employing starch-based carriers withpenetration resistance values of 25-120N were considered acceptable,with values of 30-90N representing a more preferable range and valuesbetween 40-65 N being even more preferable.

A 1″ diameter×9″ long threaded (14 tpi) push rod was mounted to theactuator of a MTS 858 Bionix Test System fitted with a 1 kN forcetransducer. A piece of 1.5″ diameter×6″ length PVC pipe was centeredvertically on the force transducer and a large weigh boat was placedunderneath it to catch the extruded bone formulation. A 1″I.D..times.0.5″ thick spacer was placed on top of the PVC tubing and7.00 g of bone mixture was weighed into a 5 cc syringe and loaded intothe tip of the syringe using a clean, dry 5 cc syringe plunger with thetip removed just below the o-ring to create a flat surface. The loadedsyringe was placed vertically into the spacer/PVC pipe assembly with theplunger facing up. The whole assembly (PVC pipe, spacer, and syringe)was centered on the load cell directly under the push rod. The center ofthe plunger was lined up with the center of the push rod. The 1 kN loadrange was used for the first test of each new bone formulation. When themaximum load required to extrude the bone mixture was less than 90N,then the 100N load range was used during subsequent tests to achieve ahigher degree of accuracy. The test sample was preloaded under loadcontrol to 5N, the displacement was zeroed, and the test was executed.Bone formulations were extruded at a rate of 5 mm/min to a maximumdisplacement of 20 mm in compression. The average maximum force requiredto extrude each bone formulation was then determined.

Example 20 Detection of Amylase Sensitivity

This example describes the assessment of amylase resistance forstarch-based stabilizers and diffusion barriers (carriers). Increasingthe amylase resistance of a starch-based carrier increases the effectiveresidence time of the carrier following implantation and thereforeenhances the stabilizing effect of the carrier.

Quantification of resistant starches requires the use of pancreatic aamylase and amyloglucosidase that effectively detect the breakdown ofamylase-resistant starches to glucose.

The breakdown of the starch and starch/lipid compositions of Examples 3,9, 15, and 17 as well as new candidate amylase resistant starches andmodified starches, is monitored using the resistant starch assay kitfrom Megazyme International Ireland Ltd. (Amyloglucosidasealpha.-Amylase Method AOAC Method 996.11, AACC Method 76.13, ICCStandard Method No. 168). Formulations with slowest breakdown willgenerally have the longest stabilization effect in vivo.

Example 21 Starch/Lipid Carrier Compositions

The following compositions were prepared in a similar fashion to thosedescribed in Examples 3, 9, 15, and 17. Carriers were autoclaved for 20minutes to sterilize them prior to mixing with DBM.

Combination #1—Carrier 1 consisted of about 8% Penford Maps 281 and 5%Lecithin with the remainder being water.

Combination #2—Carrier 2 consisted of about 8% Penford Maps 281 and 15%Lecithin with the remainder being water.

Combination #3—Carrier 3 consisted of about 6% GPC B980 and 5% Lecithinwith the remainder being water.

Combination #4—Carrier 4 consisted of about, 6% GPC B980 and 15%Lecithin with the remainder being water.

Each of the four carrier combinations were mixed with human DBM to yielda bone content of about 25%. These bone mixtures were then tested forosteoinductivity as previously described in Example 6.

Other Embodiments

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

1-105. (canceled)
 106. A demineralized bone matrix composition forimplantation in a body, the composition comprising: emineralized bonematrix and an excipient, wherein the composition exhibits anosteoinductivity that is at least about 10% greater thanosteoinductivity of a composition of demineralized bone matrix withoutexcipient.
 107. The composition of claim 106, wherein the demineralizedbone matrix comprises particles of at least 1 mm in their largestdimension.
 108. The composition of claim 106, wherein the demineralizedbone matrix comprises particles of at least 1.5 mm in their largestdimension.
 109. The composition of claim 106, wherein the demineralizedbone matrix comprises particles of at least 2 mm in their largestdimension.
 110. The composition of claim 106, wherein the demineralizedbone matrix comprises particles of at least 100 microns in theirsmallest dimension.
 111. The composition of claim 106, wherein thedemineralized bone matrix comprises particles of at least 500 microns intheir smallest dimension.
 112. The composition of claim 106, wherein thedemineralized bone matrix comprises particles of at least 800 microns intheir smallest dimension.
 113. The composition of claim 48, wherein thedemineralized bone matrix comprises particles: wherein the particles aretapered, wedge-shaped, or cone-shaped; and wherein the particles are atleast about 1 mm in their largest dimension and wherein the particlesare approximately 100 microns in another dimension.
 114. The compositionof claim 106, wherein the composition exhibits an osteoinductivity thatis at least about 20% greater than osteoinductivity of a composition ofdemineralized bone matrix without excipient.
 115. The composition ofclaim 106, wherein the composition exhibits an osteoinductivity that isat least about 35% greater than osteoinductivity of a composition ofdemineralized bone matrix without excipient.
 116. The composition ofclaim 106, wherein the excipient is selected from the group consistingof diffusion barriers, enzyme inhibitors, competitive substrates,masking entities, and combinations thereof.
 117. The composition ofclaim 106, wherein the excipient is selected from the group consistingof natural polymers, non-natural polymers, modified or derivatizednatural polymers, modified or derivated non-natural polymers, andcombinations thereof, and wherein the demineralized bone matrix is atleast partially embedded within the excipient.
 118. The composition ofclaim 117, wherein the excipient is selected from the group consistingof polysaccharides, lipids, and combinations thereof.
 119. Thecomposition of claim 117, wherein the excipient is alipid/polysaccharide combination.
 120. The composition of claim 119,wherein the lipid is phosphatidylcholine and the starch is amylaseresistant starch.
 121. The composition of claim 106, whereinosteoinductivity is at least 1 on a scale of 1 to 4 at least 30 daysfollowing implantation.
 122. The composition of claim 106, whereinosteoinductivity is at least 2 on a scale of 1 to 4 at least 30 daysfollowing implantation.
 123. The composition of claim 106, whereinosteoinductivity is at least 3 on a scale of 1 to 4 at least 30 daysfollowing implantation.
 124. The composition of claim 106, whereinosteoinductivity is at least 4 on a scale of 1 to 4 at least 30 daysfollowing implantation.
 125. The composition of claim 106, whereinosteoinductivity is at least 1 on a scale of 1 to 4 at least 45 daysfollowing implantation.
 126. The composition of claim 106, whereinosteoinductivity is at least 2 on a scale of 1 to 4 at least 45 daysfollowing implantation.
 127. The composition of claim 106, whereinosteoinductivity is at least 3 on a scale of 1 to 4 at least 45 daysfollowing implantation.
 128. The composition of claim 106, whereinosteoinductivity is at least 4 on a scale of 1 to 4 at least 45 daysfollowing implantation.
 129. The composition of claim 106, whereinosteoinductivity is at least 1 on a scale of 1 to 4 at least 60 daysfollowing implantation.
 130. The composition of claim 106, whereinosteoinductivity is at least 2 on a scale of 1 to 4 at least 60 daysfollowing implantation.
 131. The composition of claim 1068, whereinosteoinductivity is at least 3 on a scale of 1 to 4 at least 60 daysfollowing implantation.
 132. The composition of claim 48, whereinosteoinductivity is at least 4 on a scale of 1 to 4 at least 60 daysfollowing implantation.
 133. A demineralized bone matrix composition forimplantation in a body, the composition comprising demineralized bonematrix and an excipient, the composition exhibiting an osteoinductivityof at least about 25% of an osteoinductivity of a 10 μg BMP-collagensponge preparation.
 134. The composition of claim 133, wherein thedemineralized bone matrix comprises particles of at least 1 mm in theirlargest dimension.
 135. The composition of claim 133, wherein thedemineralized bone matrix comprises particles of at least 100 microns intheir smallest dimension.
 136. The composition of claim 133, wherein thedemineralized bone matrix comprises particles of at least 500 microns intheir smallest dimension.
 137. The composition of claim 133, wherein thedemineralized bone matrix comprises particles of at least 800 microns intheir smallest dimension.
 138. The composition of claim 133, wherein theexcipient is selected from the group consisting of diffusion barriers,enzyme inhibitors, competitive substrates, masking entities, andcombinations thereof.
 139. The composition of claim 133, wherein theexcipient is selected from the group consisting of natural polymers,non-natural polymers, modified or derivatized natural polymers, modifiedor derivated non-natural polymers, and combinations thereof, and whereinthe demineralized bone matrix is at least partially embedded within theexcipient.
 140. An implantable bone growth inducing compositioncomprising: a matrix; at least one growth factor; and a stabilizingagent wherein the stabilizing agent enhances the osteoinductivity of thecomposition resulting in improved bone formation ability as compared tothe composition without the stabilizing agent, wherein the growth factoris selected from the group consisting of osteogenic factors,vascularizing factors, angiogenic factors, and combinations thereof.